U.S. patent number 9,637,557 [Application Number 13/092,708] was granted by the patent office on 2017-05-02 for production of heteromultimeric proteins.
This patent grant is currently assigned to Genentech, Inc.. The grantee listed for this patent is Justin Scheer, Christoph Spiess, Daniel G. Yansura. Invention is credited to Justin Scheer, Christoph Spiess, Daniel G. Yansura.
United States Patent |
9,637,557 |
Scheer , et al. |
May 2, 2017 |
**Please see images for:
( Certificate of Correction ) ** |
Production of heteromultimeric proteins
Abstract
Described herein are methods for the efficient production of
antibodies and other multimeric protein complexes (collectively
referred to herein as heteromultimeric proteins) capable of
specifically binding to more than one target. The targets may be,
for example, different epitopes on a single molecule or located on
different molecules. The methods combine efficient, high gene
expression level, appropriate assembly, and ease of purification
for the heteromultimeric proteins. The invention also provides
methods of using these heteromultimeric proteins, and compositions,
kits and articles of manufacture comprising these antibodies.
Inventors: |
Scheer; Justin (San Francisco,
CA), Spiess; Christoph (Los Altos, CA), Yansura; Daniel
G. (Pacifica, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Scheer; Justin
Spiess; Christoph
Yansura; Daniel G. |
San Francisco
Los Altos
Pacifica |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
Genentech, Inc. (South San
Francisco, CA)
|
Family
ID: |
44121029 |
Appl.
No.: |
13/092,708 |
Filed: |
April 22, 2011 |
Prior Publication Data
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|
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Document
Identifier |
Publication Date |
|
US 20110287009 A1 |
Nov 24, 2011 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61327302 |
Apr 23, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/244 (20130101); A61P 29/00 (20180101); C07K
16/468 (20130101); C07K 16/2863 (20130101); A61P
37/08 (20180101); C07K 16/247 (20130101); A61P
37/02 (20180101); C07K 16/30 (20130101); A61P
35/00 (20180101); C07K 2317/31 (20130101); A61K
2039/505 (20130101); C07K 2317/92 (20130101); C07K
2317/90 (20130101); C07K 2317/73 (20130101); C07K
2317/76 (20130101); C07K 2317/526 (20130101); Y02A
50/30 (20180101) |
Current International
Class: |
C07K
16/00 (20060101); C07K 16/46 (20060101); C07K
16/28 (20060101); C07K 16/30 (20060101); C07K
16/24 (20060101); A61K 39/00 (20060101) |
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|
Primary Examiner: Duffy; Brad
Attorney, Agent or Firm: Morrison & Foerster LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Patent
Application Ser. No. 61/327,302, entitled "Production of
Heteromultimeric Proteins," filed 23 Apr. 2010, the entire contents
of which are incorporated herein by reference.
Claims
What is claimed is:
1. A method of preparing a heteromultimeric protein comprising a
first antibody heavy chain paired with a first antibody light chain
and a second antibody heavy chain paired with a second antibody
light chain, and wherein the first and second antibody heavy chains
are linked by at least one interchain disulfide bond, the method
comprising the steps of: (a) culturing a first host cell comprising
a first nucleic acid encoding the first antibody heavy chain and
the first antibody light chain under conditions where a first half
antibody comprising the first antibody heavy chain and the first
antibody light chain is expressed; (b) culturing a second host cell
comprising a second nucleic acid encoding the second antibody heavy
chain and the first antibody light chain under conditions where a
second half antibody comprising the second antibody heavy chain and
the second antibody light chain is expressed; (c) combining
cultures of the first and second host cells to produce a combined
culture comprising the first host cells and second host cells,
wherein the heteromultimeric protein is formed in the combined
culture; and (d) recovering the heteromultimeric protein without
prior recovery or purification of the first and second half
antibodies from separate cultures.
2. The method of claim 1, wherein the first antibody heavy chain
comprises a first heterodimerization domain and the second antibody
heavy chain comprises a second heterodimerization domain.
3. The method of claim 1, wherein the first antibody heavy chain
comprises a first Fc domain and the second antibody heavy chain
comprises a second Fc domain.
4. The method of claim 3, wherein the first Fc domain and the
second Fc domain meet at an interface, wherein the interface of the
first Fc domain comprises a cavity, and wherein the interface of
the second Fc domain comprises a protuberance which is positionable
in the cavity in the interface of the first Fc domain.
5. The method of claim 2, wherein the first heterodimerization
domain and the second heterodimerization domain comprise a leucine
zipper.
6. The method of claim 2, wherein the first heterodimerization
domain and the second heterodimerization domain comprise a coiled
coil.
7. The method of claim 1, wherein said at least one interchain
disulfide bond is in the hinge region of an antibody.
8. The method of claim 1, wherein said at least one interchain
disulfide bond is between hinge regions of the first and second
antibody heavy chains.
9. The method of claim 1, wherein the first and/or second antibody
heavy chain comprises a C.sub.H-domain or variant thereof.
10. The method of claim 1, wherein the first and/or second antibody
heavy chain comprises an Fc region or variant thereof.
11. The method of claim 1, wherein the first antibody heavy chain
and the first antibody light chain polypeptide form a first target
binding arm, and wherein the second antibody heavy chain and the
second antibody light chain form a second target binding arm.
12. The method of claim 1, wherein the first and second antibody
heavy chains each comprise at least a portion of a human C.sub.H2
and/or C.sub.H3 domain.
13. The method of claim 1, wherein the first and second half
antibodies are humanized.
14. The method of claim 1, wherein the first antibody heavy chain
and first antibody light chain are encoded on separate expression
plasmids.
15. The method of claim 1, wherein the first antibody heavy chain
and first antibody light chain are encoded on the same expression
plasmid.
16. The method of claim 1, wherein the first and second host cells
are selected from the group consisting of prokaryotic cells,
eukaryotic cells, mammalian cells and plant cells.
17. The method of claim 1, wherein the first and second host cells
are prokaryotic cells.
18. The method of claim 17, wherein said prokaryotic cells are E.
coli cells.
19. The method of claim 18, wherein said E. coli cells are lpp
deficient.
20. The method of claim 1, wherein the first and second host cells
are mammalian cells.
21. The method of claim 20, wherein said mammalian cells are CHO
cells.
22. The method of claim 1, wherein the recovery step further
comprises at least one purification step.
23. The method of claim 22, wherein the at least one purification
step comprises: (i) capturing said heteromultimeric protein on a
column comprising Protein A, (ii) eluting said heteromultimeric
protein from said column, and (iii) diluting said eluted
heteromultimeric protein into a solution containing a chaotropic
agent or mild detergent.
24. The method of claim 1, wherein the heteromultimeric protein is
selected from the group consisting of an antibody, a bispecific
antibody, a multispecific antibody, a one-armed antibody, a
multispecific monovalent antibody, a bispecific maxibody, a
monobody, an immunoadhesin, a peptibody, a bispecific peptibody, a
monovalent peptibody, and an affibody.
25. The method of claim 24, wherein the heteromultimeric protein is
an antibody.
26. The method of claim 24, wherein the heteromultimeric protein is
a bispecific antibody.
27. The method of claim 24, wherein the heteromultimeric protein is
a multispecific antibody.
28. The method of claim 24, wherein the heteromultimeric protein is
a one-armed antibody.
29. The method of claim 25, wherein said antibody comprises a heavy
chain constant domain and a light chain constant domain.
30. The method of claim 25, wherein said antibody is humanized.
31. The method of claim 25, wherein the antibody is a full-length
antibody.
32. The method of claim 1, wherein the first antibody heavy chain
and first antibody light chain are human.
33. The method of claim 25, wherein said antibody is human.
34. The method of claim 25, wherein the antibody is an antibody
fragment comprising at least a portion of human C.sub.H2 and/or
C.sub.H3 domain.
35. The method of claim 34, wherein said antibody fragment is an Fc
fusion polypeptide.
36. The method of claim 26, wherein the bispecific antibody is
selected from the group consisting of IgG, IgA and IgD.
37. The method of claim 36, wherein the bispecific antibody is
IgG.
38. The method of claim 37, wherein the IgG is IgG1.
39. The method of claim 37, wherein the IgG is IgG2.
40. The method of claim 25, wherein the antibody is a therapeutic
antibody.
41. The method of claim 25, wherein the antibody is an agonist
antibody.
42. The method of claim 25, wherein the antibody is an antagonistic
antibody.
43. The method of claim 25, wherein the antibody is a diagnostic
antibody.
44. The method of claim 25, wherein the antibody is a blocking
antibody.
45. The method of claim 25, wherein the antibody is a neutralizing
antibody.
46. The method of claim 25, wherein the antibody is capable of
binding to a tumor antigen.
47. The method of claim 46, wherein the tumor antigen is not a cell
surface molecule.
48. The method of claim 46, wherein the tumor antigen is not a
cluster differentiation factor.
49. The method of claim 25, wherein the antibody is capable of
binding to a cluster differentiation factor.
50. The method of claim 25, wherein the antibody is capable of
binding to a cell survival regulatory factor.
51. The method of claim 25, wherein the antibody is capable of
binding specifically to a cell proliferation regulatory factor.
52. The method of claim 25, wherein the antibody is capable of
binding to a molecule associated with tissue development or
differentiation.
53. The method of claim 25, wherein the antibody is capable of
binding to a cell surface molecule.
54. The method of claim 25, wherein the antibody is capable of
binding to a lymphocyte.
55. The method of claim 26, wherein the first and second light
chains of the bispecific antibody comprise different variable
domain sequences.
56. The method of claim 39, wherein at least 40% of the first
half-antibody and second half-antibody form said bispecific
antibody.
57. The method of claim 26, wherein said bispecific antibody is
capable of specifically binding two different antigens or two
epitopes on the same antigen.
58. The method of claim 57, wherein the bispecific antibody is
capable of specifically binding two different antigens.
59. The method of claim 1, wherein at least 40% of the first half
antibody and second half antibody in the combined culture form the
heteromultimeric protein.
60. The method of claim 22, wherein no more than 10% of the first
and second half antibodies are present as monomers or homodimers
prior to the step of purifying the heteromultimeric protein.
61. The method of claim 1, wherein the first and second antibody
heavy chains comprise different variable domain sequences and
wherein the first and second antibody light chains comprise
different variable domain sequences.
62. The method of claim 1, wherein the first antibody heavy chain
and first antibody light chain are linked to each other via
disulfide bonds, and wherein the second antibody heavy chain and
second antibody light chain are linked to each other via disulfide
bonds.
63. The method of claim 1, wherein the difference in pI values
between the first half antibody and second half antibody is at
least 0.5.
64. The method of claim 1, further comprising a step of adding a
reductant.
65. The method of claim 1, wherein the first and second half
antibodies are secreted into culture medium of the combined culture
comprising the first host cells and second host cells.
66. The method of claim 65, wherein the heteromultimeric protein is
formed in the culture medium of the combined culture comprising the
first host cells and second host cells.
67. The method of claim 1, further comprising disrupting the cell
membrane in the combined culture comprising the first host cells
and second host cells to form the heteromultimeric protein.
68. The method of claim 67, wherein disrupting the cell membrane is
selected from the group consisting of permeabilizing the cell
membrane and cell membrane disintegration.
69. The method of claim 68, wherein disrupting the cell membrane is
selected from the group consisting of enzymatic lysis, cell lysis,
sonication, osmotic shock, passage through a microfluidizer,
addition of EDTA, various detergents, solvents (such as toluene,
dimethyl sulfoxide, etc), surfactants (such as Triton-X 100, Tween
20, etc), hypotonic buffers, use of freeze/thaw techniques,
electroporation, and passage through a stainless steel ball
homogenizer.
70. The method of claim 1, wherein the first and second nucleic
acids are recombinant nucleic acids.
71. The method of claim 3, wherein the first Fc domain and the
second Fc domain meet at an interface, wherein the interface of the
second Fc domain comprises a cavity, and wherein the interface of
the first Fc domain comprises a protuberance which is positionable
in the cavity in the interface of the second Fc domain.
72. The method of claim 1, wherein the second antibody heavy chain
and second antibody light chain are encoded on separate expression
plasmids.
73. The method of claim 1, wherein the second antibody heavy chain
and second antibody light chain are encoded on the same expression
plasmid.
74. The method of claim 1, wherein the second antibody heavy chain
and second antibody light chain are human.
75. The method of claim 67, wherein the first and second host cells
are prokaryotic cells.
Description
TECHNICAL FIELD
This invention relates to methods for the production of
heteromultimeric proteins.
BACKGROUND
Monoclonal antibodies of the IgG type contain two identical
antigen-binding arms and a constant domain (Fc). Antibodies with a
differing specificity in their binding arms usually do not occur in
nature and, therefore, have to be crafted with the help of chemical
engineering (e.g., chemical cross-linking, etc), recombinant DNA
and/or cell-fusion technology.
Bispecific antibodies can bind simultaneously two different
antigens. This property enables the development of therapeutic
strategies that are not possible with conventional monoclonal
antibodies. The large panel of imaginative bispecific antibody
formats that has been developed reflects the strong interest for
these molecules. See Berg J, Lotscher E, Steimer K S, et al.,
"Bispecific antibodies that mediate killing of cells infected with
human immunodeficiency virus of any strain," Proc Natl Acad Sci USA
(1991) 88(11): 4723-4727 and Fischer N and Leger O., "Biospecific
Antibodies: Molecules That Enable Novel Therapeutic Strategies,"
Pathobiology (2007) 74:3-14.
Another class of multispecific molecules is recombinant fusion
proteins. Recombinant fusion proteins consisting of the
extracellular domain of immunoregulatory proteins and the constant
(Fc) domain of immunoglobulin (Ig) represent a growing class of
human therapeutics. Immunoadhesins combine the binding region of a
protein sequence, with a desired specificity, with the effector
domain of an antibody. Immunoadhesins have two important properties
that are significant to their potential as therapeutic agents: the
target specificity, and the pharmacokinetic stability (half-life in
vivo that is comparable to that of antibodies). Immunoadhesins can
be used as antagonist to inhibit or block deleterious interactions
or as agonist to mimic or enhance physiological responses. See
Chamow S M, Zhang D Z, Tan X Y, et al., "A humanized, bispecific
immunoadhesin-antibody that retargets CD3+ effectors to kill
HIV-1-infected cells," J Hematother 1995; 4(5): 439-446.
Other multispecific molecules have been discussed elsewhere.
Examples include but are not limited to: Fisher et al.,
Pathobiology (2007) 74:3-14 (review of various bispecific formats);
U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et
(peptibodies); US Pat. Publ. No. 2002-004587 published Jan. 10,
2002 (multispecific antibodies); U.S. Pat. No. 7,612,181 issued
Nov. 3, 2009 to Wu et al. (Dual Variable Domain format); U.S. Pat.
No. 6,534,628, Nord K et al., Prot Eng (1995) 8:601-608, Nord K et
al., Nat Biotech (1997) 15:772-777, and Gronwall et al., Biotechnol
Appl Biochem. (2008) June; 50(Pt 2): 97-112 (Affibodies); Martens
et al., Clin Cancer Res (2006), 12: 6144-6152 and Jin et al.,
Cancer Res (2008) 68(11):4360-4368 (one armed antibodies); Bostrom
et al., Science (2009) 323:1610-1614 (Dual Action Fab, aka mixed
valency antibodies). Other formats are known to those skilled in
the art.
The manufacturing of clinical grade material remains challenging
for the multispecific molecules described above. As noted above,
there are many paths to the production of molecules with mixed
binding arms, i.e., binding arms that are not identical to each
other. Each of these methods has its drawbacks.
Chemical cross-linking is labor intensive as the relevant species
may yet need to be purified from homodimers and other undesired
by-products. In addition, the chemical modification steps can alter
the integrity of the proteins thus leading to poor stability. Thus,
this method is often inefficient and can lead to loss of antibody
activity.
Cell-fusion technology (e.g., hybrid hybridomas) express two heavy
and two light chains that assemble randomly leading to the
generation of 10 antibody combinations. The desired
heteromultimeric antibodies are only a small fraction of the
antibodies thus produced. Purification of the desired
heteromultimeric proteins dramatically reduces production yields
and increases manufacturing costs.
Recombinant DNA techniques have been used to generate various
heteromultimeric formats, e.g., single chain Fv, diabodies, etc.,
that do not comprise an Fc domain. A major drawback for this type
of antibody molecule is the lack of the Fc domain and thus the
ability of the antibody to trigger an effector function (e.g.,
complement activation, Fc-receptor binding etc.). Thus, a
bispecific antibody comprising a functional Fc domain is
desired.
Recombinant DNA techniques have also been used to generate `knob
into hole` bispecific antibodies. See US Patent Application
20030078385 (Arathoon et al.--Genentech). One constraint of this
strategy is that the light chains of the two parent antibodies have
to be identical to prevent mispairing and formation of undesired
and/or inactive molecules due to being expressed in the same
cell.
Thus, there remains a need for alternative methods of producing
heteromultimeric proteins. The invention described herein provides
such methods. These and other aspects and advantages of the
invention will be apparent from the description of the invention
provided herein.
BRIEF SUMMARY OF THE INVENTION
Production of heteromultimeric proteins, e.g., multispecific
antibodies, using current techniques has drawbacks including the
production of a mixture of products, reduced yield and
decreased/elimination of effector function among others. Thus, it
is desirable to produce heteromultimeric proteins efficiently and
at high levels.
The production of antibody molecules, by various means, is
generally well understood. U.S. Pat. No. 6,331,415 (Cabilly et
al.), for example, describes a method for the recombinant
production of immunoglobulin where the heavy and light chains are
expressed simultaneously from a single vector or from two separate
vectors in a single cell. Wibbenmeyer et al., (1999, Biochim
Biophys Acta 1430(2): 191-202) and Lee and Kwak (2003, J.
Biotechnology 101:189-198) describe the production of monoclonal
antibodies from separately produced heavy and light chains, using
plasmids expressed in separate cultures of E. coli. Various other
techniques relevant to the production of antibodies are described
in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988)
and WO2006028936. Yet each of these have draw backs such as low
yield, use of chemicals
The inventive methods provide for the expression of each component,
e.g., one arm of an antibody, of a hinge-containing
heteromultimeric protein in a separate host cell and the assembly
of the hinge-containing heteromultimeric protein, e.g., a
multispecific antibody, without the addition of a reductant.
This invention provides an easy and efficient production
process/method that allows for the economical production of
heteromultimeric proteins, e.g., multispecific antibodies.
The invention provides efficient and novel methods of producing
multispecific immunoglobulin complexes (e.g., multispecific
antibodies) and other multimeric proteins (collectively referred to
herein as heteromultimeric proteins) that overcome limitations of
traditional methods. Heteromultimeric proteins, such as bispecific
antibodies, can be provided as a highly homogeneous heteromultimer
polypeptide according to methods of the invention. In addition, the
methods provided for herein do not rely on the addition of a
reductant to achieve the formation of at least one, at least two,
at least three, at least four interchain disulfide bonds in the
heteromultimeric protein.
In a first aspect, the method described herein allows for the
preparation of a heteromultimeric protein comprising a first
hinge-containing polypeptide having a first heterodimerization
domain and a second hinge-containing polypeptide having a second
heterodimerization domain, wherein the second heterodimerization
domain interacts with the first heterodimerization domain, and
wherein the first and second hinge-containing polypeptides are
linked by at least one interchain disulfide bond, the method
comprising the steps of: (a) culturing a first host cell comprising
a first nucleic acid encoding the first hinge-containing
polypeptide under conditions where the hinge-containing polypeptide
is expressed; (b) culturing a second host cell comprising a nucleic
acid encoding the second hinge-containing polypeptide under
conditions where the hinge-containing polypeptide is expressed; (c)
disrupting the cell membranes so that the first and second
hinge-containing polypeptides are released into the extracellular
milieu, wherein the first and second host cells have been combined
together in a single suspension; and (d) recovering the
heteromultimeric protein, wherein said method does not require the
addition of a reductant.
In a second aspect, the method of preparing a heteromultimeric
protein comprising heteromultimeric protein comprising a first
hinge-containing polypeptide having a first heterodimerization
domain and a second hinge-containing polypeptide having a second
heterodimerization domain, wherein the second heterodimerization
domain interacts with the first heterodimerization domain, and
wherein the first and second hinge-containing polypeptides are
linked by at least one interchain disulfide bond, the method
comprising the steps of: (a) providing a purified first
hinge-containing polypeptide having a first heterodimerization
domain; (b) providing a purified second hinge-containing
polypeptide having a second heterodimerization domain; (c)
combining the first and second hinge-containing polypeptides; (d)
refolding the first hinge-containing polypeptide with the second
hinge-containing polypeptide; and (e) recovering the
heteromultimeric protein complex.
In a third aspect, the methods provided for herein are directed to
a method of preparing a heteromultimeric protein comprising
incubating a first pair of immunoglobulin heavy and light chain
polypeptides, and a second pair of immunoglobulin heavy and light
chain polypeptides, under conditions permitting multimerization of
the first and second pair of polypeptides to form a substantially
homogeneous population of antibodies, wherein the conditions do not
comprise the addition of a reductant; wherein the first pair of
polypeptides is capable of binding a first target; wherein the
second pair of polypeptides is capable of binding a second target
molecule; and wherein Fc polypeptide of the first heavy chain
polypeptide and Fc polypeptide of the second heavy chain
polypeptide meet at an interface, and the interface of the second
Fc polypeptide comprises a protuberance which is positionable in a
cavity in the interface of the first Fc polypeptide.
In a fourth aspect, there is a method of generating a combinatorial
library of heteromultimeric proteins, said method comprising a
first hinge-containing polypeptide having a first
heterodimerization domain and a second hinge-containing polypeptide
having a second heterodimerization domain, wherein the second
heterodimerization domain interacts with the first
heterodimerization domain, and wherein the first and second
hinge-containing polypeptides are linked by at least one interchain
disulfide bond, the method comprising the steps of: (a) culturing a
first host cell and at least two additional host cells, wherein a.
said first host cell comprises a first nucleic acid encoding a
first heterodimerization domain-containing polypeptide; and b. said
additional host cells comprise a nucleic acid comprising a second
heterodimerization domain-containing polypeptide, (b) combining the
first and at least two additional host cells; (c) treating the
cells so that the first and second heterodimerization
domain-containing polypeptides are released into the extracellular
milieu; and (d) recovering the heteromultimeric proteins, wherein
said method does not require the addition of a reductant.
In a fifth aspect, there are provided the heteromultimeric proteins
produced by the methods described herein.
It is to be understood that methods of the invention can include
other steps which generally are routine steps evident for
initiating and/or completing the process encompassed by methods of
the invention as described herein. For example, in one embodiment,
step (a) of a method of the invention is preceded by a step wherein
a nucleic acid encoding a first hinge-containing polypeptide is
introduced into a first host cell, and a nucleic acid encoding a
second hinge-containing polypeptide is introduced into a second
host cell. In one embodiment, methods of the invention further
comprise a step of purifying heteromultirneric proteins having
binding specificity to at least two distinct targets. In one
embodiment, no more than about 10%, 15%, or 20% of isolated
polypeptides are present as monomers or heavy-light chain dimers
prior to the step of purifying the heteromultimeric proteins.
In an embodiment, the first and/or second hinge-containing
polypeptide is an antibody heavy chain. In a further embodiment,
the antibody heavy chain is paired with an antibody light chain to
provide a heavy-light chain pair. In some embodiments, the
heavy-light chain pair are covalently linked. In another
embodiment, the heavy-light chain pair defines a target binding
arm. In some embodiments, the target binding arms are identical. In
some embodiments, the target binding arms each recognize two
distinct targets.
In some embodiments, the first and/or second hinge-containing
polypeptide comprises an Fc region. In another embodiment the first
and/or second hinge-containing polypeptide comprises at least one
constant heavy domain. In another embodiment, the first and/or
second hinge-containing polypeptide comprises a variable heavy
chain domain. In another embodiment, the first and/or second
hinge-containing polypeptide comprises a receptor binding domain.
In some embodiments, the first and/or second hinge-containing
polypeptide are substantially identical (i.e., the
heterodimerization domain may not be identical with the regions
outside of the heterodimerization domain being identical). In some
embodiments, the first and/or second hinge-containing polypeptide
are not identical.
In some embodiments, the heteromultimeric protein is selected from
the group consisting of an antibody, a bispecific antibody, a
multispecific antibody, one-armed antibody, monospecific monovalent
antibody, a multispecific monovalent antibody, a bispecific
maxibody, a monobody, an immunoadhesin, a peptibody, a bispecific
peptibody, a monovalent peptibody, an affibody and a receptor
fusion protein.
In some embodiments, said heteromultimeric proteins comprise a
hinge region that has at least one, at least two, at least three,
at least four, or any integer number up to all, of the cysteine
residues that are normally capable of forming an inter-heavy chain
disulfide linkage. In some embodiments, additional cysteines have
been introduced into the hinge region.
A heteromultimeric protein of the invention may also be an antibody
fragment, such as, for example, an Fc or Fc fusion polypeptide, so
long as it comprises the hinge region of an immunoglobulin. An Fc
fusion polypeptide generally comprises an Fc polypeptide (or
fragment thereof) fused to a heterologous polypeptide sequence
(such as an antigen binding domain), such as a receptor
extracellular domain (ECD) fused to an immunoglobulin Fc
polypeptide (e.g., Flt receptor ECD fused to a IgG2 Fc). For
example, in one embodiment, an Fc fusion polypeptide comprises a
VEGF binding domain, which may be a VEGF receptor, which includes
flt, flk, etc. A heteromultimeric protein of the invention
generally comprises a heavy chain constant domain and a light chain
constant domain. In one embodiment, a heteromultimeric protein of
the invention comprises a modification (for example, but not
limited to, insertion of one or more amino acids, e.g., to form a
dimerization sequence such as leucine zipper) for formation of
inter-heavy chain dimerization or multimerization. In some
embodiments, a portion (but not all) of the Fc polypeptide is
missing in a heteromultimer of the invention, so long as it retains
the hinge region of an immunoglobulin. In some of these
embodiments, the missing sequence of the Fc polypeptide is a
portion or all of the C.sub.H2 and/or C.sub.H3 domain. In some of
these embodiments, the heteromultimeric protein comprises a
dimerization domain (such as a leucine zipper sequence), for
example fused to the C-terminus of the heavy chain fragment. In
some of these embodiments, the heteromultimeric protein comprises a
dimerization domain comprising mutations to provide for a "knob
into hole" dimerization domain (as further defined below).
In some embodiments of the methods and heteromultimeric proteins of
the invention, the hinge-containing polypeptides comprise at least
one characteristic that promotes heterodimerization, while
minimizing homodimerization, of the first and second
hinge-containing polypeptides (e.g., between Fc polypeptides of the
heavy chains). Such characteristic(s) improves yield and/or purity
and/or homogeneity of the heteromultimeric protein populations
obtainable by methods of the invention as described herein. In one
embodiment, the Fc polypeptides of a first hinge-containing
polypeptide and a second hinge-containing polypeptide meet/interact
at an interface. In some embodiments wherein the Fc polypeptides of
the first and second hinge-containing polypeptides meet at an
interface, the interface of the second Fc polypeptide comprises a
protuberance which is positionable in a cavity in the interface of
the first Fc polypeptide. In one embodiment, the first Fc
polypeptide has been altered from a template/original polypeptide
to encode the cavity or the second Fc polypeptide has been altered
from a template/original polypeptide to encode the protuberance, or
both. In one embodiment, the first Fc polypeptide has been altered
from a template/original polypeptide to encode the cavity and the
second Fc polypeptide has been altered from a template/original
polypeptide to encode the protuberance, or both. In one embodiment,
the interface of the second Fc polypeptide comprises a protuberance
which is positionable in a cavity in the interface of the first Fc
polypeptide, wherein the cavity or protuberance, or both, have been
introduced into the interface of the first and second Fc
polypeptides, respectively. In some embodiments wherein the first
and second Fc polypeptides meet at an interface, the interface of
the first Fc polypeptide comprises a protuberance which is
positionable in a cavity in the interface of the second Fc
polypeptide. In one embodiment, the second Fc polypeptide has been
altered from a template/original polypeptide to encode the cavity
or the first Fc polypeptide has been altered from a
template/original polypeptide to encode the protuberance, or both.
In one embodiment, the second Fc polypeptide has been altered from
a template/original polypeptide to encode the cavity and the first
Fc polypeptide has been altered from a template/original
polypeptide to encode the protuberance, or both. In one embodiment,
the interface of the first Fc polypeptide comprises a protuberance
which is positionable in a cavity in the interface of the second Fc
polypeptide, wherein the protuberance or cavity, or both, have been
introduced into the interface of the first and second Fc
polypeptides, respectively.
In one embodiment, the protuberance and cavity each comprises a
naturally occurring amino acid residue. In one embodiment, the Fc
polypeptide comprising the protuberance is generated by replacing
an original residue from the interface of a template/original
polypeptide with an import residue having a larger side chain
volume than the original residue. In one embodiment, the Fc
polypeptide comprising the protuberance is generated by a method
comprising a step wherein nucleic acid encoding an original residue
from the interface of said polypeptide is replaced with nucleic
acid encoding an import residue having a larger side chain volume
than the original. In one embodiment, the original residue is
threonine. In one embodiment, the import residue is arginine (R).
In one embodiment, the import residue is phenylalanine (F). In one
embodiment, the import residue is tyrosine (Y). In one embodiment,
the import residue is tryptophan (W). In one embodiment, the import
residue is R, F, Y or W. In one embodiment, a protuberance is
generated by replacing two or more residues in a template/original
polypeptide. In one embodiment, the Fc polypeptide comprising a
protuberance comprises replacement of threonine at position 366
with tryptophan, amino acid numbering according to the EU numbering
scheme of Kabat et al. (pp. 688-696 in Sequences of proteins of
immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda,
Md.)).
In some embodiments, the Fc polypeptide comprising a cavity is
generated by replacing an original residue in the interface of a
template/original polypeptide with an import residue having a
smaller side chain volume than the original residue. For example,
the Fc polypeptide comprising the cavity may be generated by a
method comprising a step wherein nucleic acid encoding an original
residue from the interface of said polypeptide is replaced with
nucleic acid encoding an import residue having a smaller side chain
volume than the original. In one embodiment, the original residue
is threonine. In one embodiment, the original residue is leucine.
In one embodiment, the original residue is tyrosine. In one
embodiment, the import residue is not cysteine (C). In one
embodiment, the import residue is alanine (A). In one embodiment,
the import residue is serine (S). In one embodiment, the import
residue is threonine (T). In one embodiment, the import residue is
valine (V). A cavity can be generated by replacing one or more
original residues of a template/original polypeptide. For example,
in one embodiment, the Fc polypeptide comprising a cavity comprises
replacement of two or more original amino acids selected from the
group consisting of threonine, leucine and tyrosine. In one
embodiment, the Fc polypeptide comprising a cavity comprises two or
more import residues selected from the group consisting of alanine,
serine, threonine and valine. In some embodiments, the Fc
polypeptide comprising a cavity comprises replacement of two or
more original amino acids selected from the group consisting of
threonine, leucine and tyrosine, and wherein said original amino
acids are replaced with import residues selected from the group
consisting of alanine, serine, threonine and valine. In one
embodiment, the Fc polypeptide comprising a cavity comprises
replacement of threonine at position 366 with serine, amino acid
numbering according to the EU numbering scheme of Kabat et al.,
supra. In one embodiment, the Fc polypeptide comprising a cavity
comprises replacement of leucine at position 368 with alanine,
amino acid numbering according to the EU numbering scheme of Kabat
et al., supra. In one embodiment, the Fc polypeptide comprising a
cavity comprises replacement of tyrosine at position 407 with
valine, amino acid numbering according to the EU numbering scheme
of Kabat et al., supra. In one embodiment, the Fc polypeptide
comprising a cavity comprises two or more amino acid replacements
selected from the group consisting of T366S, L368A and Y407V, amino
acid numbering according to the EU numbering scheme of Kabat et
al., supra. In some embodiments of these antibody fragments, the Fc
polypeptide comprising the protuberance comprises replacement of
threonine at position 366 with tryptophan, amino acid numbering
according to the EU numbering scheme of Kabat et al., supra.
In various embodiments, the Fc polypeptide of the first and second
heavy chain polypeptides may or may not be identical, provided they
are capable of dimerizing to form an Fc region (as defined herein).
A first Fc polypeptide is generally contiguously linked to one or
more domains of an immunoglobulin heavy chain in a single
polypeptide, for example with hinge, constant and/or variable
domain sequences. In one embodiment, the first Fc polypeptide
comprises at least a portion (including all) of a hinge sequence,
at least a portion (including all) of a C.sub.H2 domain and/or at
least a portion (including all) of a C.sub.H3 domain. In one
embodiment, the first Fc polypeptide comprises the hinge sequence
and the C.sub.H2 and C.sub.H3 domains of an immunoglobulin. In one
embodiment, the second Fc polypeptide comprises at least a portion
(including all) of a hinge sequence, at least a portion (including
all) of a C.sub.H2 domain and/or at least a portion (including all)
of a C.sub.H3 domain. In one embodiment, the second Fc polypeptide
comprises the hinge sequence and the C.sub.H2 and C.sub.H3 domains
of an immunoglobulin. In one embodiment, an antibody of the
invention comprises first and second Fc polypeptides each of which
comprising at least a portion of at least one antibody constant
domain. In one embodiment, the antibody constant domain is a
C.sub.H2 and/or C.sub.H3 domain. In any of the embodiments of an
antibody of the invention that comprises a constant domain, the
antibody constant domain can be from any immunoglobulin class, for
example an IgG. The immunoglobulin source can be of any suitable
species of origin (e.g., an IgG may be human IgG.sub.1) or of
synthetic form.
In one embodiment, a first light chain polypeptide and a second
light chain polypeptide in a first and second target molecule
binding arm, respectively, of an antibody of the invention comprise
different/distinct antigen binding determinants (e.g.,
different/distinct variable domain sequences). In one embodiment, a
first light chain polypeptide and a second light chain polypeptide
in a first and second target molecule binding arm, respectively, of
an antibody of the invention comprise the same (i.e., a common)
antigen binding determinant e.g., the same variable domain
sequence).
Methods of the invention are capable of generating heteromultimeric
molecules at high homogeneity. According, the invention provides
methods wherein at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of polypeptides are in
a complex comprising a first heavy and light chain polypeptide pair
and a second heavy and light chain polypeptide pair. In one
embodiment, the invention provides methods wherein between about 60
and 99%, 70 and 98%, 75 and 97%, 80 and 96%, 85 and 96%, or 90 and
95% of polypeptides are in a complex comprising a first heavy and
light chain polypeptide pair and a second heavy and light chain
polypeptide pair.
In one embodiment, an antibody of the invention is selected from
the group consisting of IgG, IgE, IgA, IgM and IgD. In some
embodiments, the hinge region of an antibody of the invention is
preferably of an immunoglobulin selected from the group consisting
of IgG, IgA and IgD. For example, in some embodiments, an antibody
or hinge region of an antibody is of IgG, which in some embodiments
is IgG1 or IgG2 (e.g., IgG2a or IgG2b). In some embodiments, an
antibody of the invention is selected from the group consisting of
IgG, IgA and IgD. In one embodiment, the antibody is of human,
humanized, chimeric or non-human (e.g., murine) origin.
Heteromultimeric proteins of the invention generally are capable of
binding, preferably specifically, to antigens. Such antigens
include, for example, tumor antigens, cell survival regulatory
factors, cell proliferation regulatory factors, molecules
associated with (e.g., known or suspected to contribute
functionally to) tissue development or differentiation, cell
surface molecules, lymphokines, cytokines, molecules involved in
cell cycle regulation, molecules involved in vasculogenesis and
molecules associated with (e.g., known or suspected to contribute
functionally to) angiogenesis. An antigen to which a
heteromultimeric protein of the invention is capable of binding may
be a member of a subset of one of the above-mentioned categories,
wherein the other subset(s) of said category comprise other
molecules/antigens that have a distinct characteristic (with
respect to the antigen of interest). An antigen of interest may
also be deemed to belong to two or more categories. In one
embodiment, the invention provides a heteromultimeric protein that
binds, preferably specifically, a tumor antigen that is not a cell
surface molecule. In one embodiment, a tumor antigen is a cell
surface molecule, such as a receptor polypeptide. In another
example, in some embodiments, a heteromultimeric protein of the
invention binds, preferably specifically, a tumor antigen that is
not a cluster differentiation factor. In another example, a
heteromultimeric protein of the invention is capable of binding,
preferably specifically, to a cluster differentiation factor, which
in some embodiments is not, for example, CD3 or CD4. In some
embodiments, a heteromultimeric protein of the invention is an
anti-VEGF antibody. In some embodiments, a heteromultimeric protein
of the invention is a bispecific antibody selected from the group
consisting of IL-1alpha/IL-1beta, IL-12/IL-18; IL-13/IL-9;
IL-13/IL-4; IL-13/IL-5; IL-5/IL-4; IL-13/IL-(beta; IL-13/IL-25;
IL-13/TARC; IL-13/MDC; IL-13/MEF; IL-13/TGF-.beta.; IL-13/LHR
agonist; IL-12/TWEAK, IL-13/CL25; IL-13/SPRR2a; IL-13/SPRR2b;
IL-13/ADAMS, IL-13/PED2, IL17A/IL17F, CD3/CD19, CD138/CD20;
CD138/CD40; CD19/CD20; CD20/CD3; CD38/CD138; CD38/CD20; CD38/CD40;
CD40/CD20; CD-8/IL-6; CD20/BR3, TNFalpha/TGF-beta, TNFalpha/IL-1
beta; TNFalpha/IL-2, TNF alpha/IL-3, TNFalpha/IL-4, TNFalpha/IL-5,
TNFalpha/IL6, TNFalpha/IL8, TNFalpha/IL-9, TNFalpha/IL-10,
TNFalpha/IL-11, TNFalpha/IL-12, TNFalpha/IL-13, TNFalpha/IL-14,
TNFalpha/IL-15, TNFalpha/IL-16, TNFalpha/IL-17, TNFalpha/IL-18,
TNFalpha/IL-19, TNFalpha/IL-20, TNFalpha/IL-23, TNFalpha/IFNalpha,
TNFalpha/CD4, TNFalpha/VEGF, TNFalpha/MIF, TNFalpha/ICAM-1,
TNFalpha/PGE4, TNFalpha/PEG2, TNFalpha/RANK ligand, TNFalpha/Te38;
TNFalpha/BAFF; TNFalpha/CD22; TNFalpha/CTLA-4; TNFalpha/GP130;
TNF.alpha./IL-12p40; VEGF/HER2, VEGF-A/HER2, VEGF-A/PDGF,
HER1/HER2, VEGF-A/VEGF-C, VEGF-C/VEGF-D, HER2/DR5, VEGF/IL-8,
VEGF/MET, VEGFR/MET receptor, VEGFR/EGFR, HER2/CD64, HER2/CD3,
HER2/CD16, HER2/HER3; EGFR/HER2, EGFR/HER3, EGFR/HER4, IL-13/CD40L,
IL4/CD40L, TNFR1/IL-1R, TNFR1/IL-6R, TNFR1/IL-18R, EpCAM/CD3,
MAPG/CD28, EGFR/CD64, CSPGs/RGM A; CTLA-4/BTNO2; IGF1/IGF2;
IGF1/2/Erb2B; MAG/RGM A; NgR/RGM A; NogoA/RGM A; OMGp/RGM A;
PDL-I/CTLA-4; and RGM A/RGM B, IL1.beta./IL18, NRP1/VEGFA,
VEGFA/NRP2, cMET/EGFR, ALK1/BMP9, VEGFA/.alpha.5.beta.1,
HER1/HER3-BU, and CMV. In some embodiments, a heteromultimeric
protein of the invention binds to at least two target molecules
selected from the group consisting of: .alpha.5.beta.1, ALK1, BMP9,
IL-1 alpha, IL-1 beta, TARC, MDC, MEF, TGF-.beta., LHR agonist,
TWEAK, CL25, SPRR2a, SPRR2b, ADAM8, PED2, CD3, CD4, CD16, CD19,
CD20, CD22, CD28, CD40, CD38, CD64, CD138, CD-8, BR3, TNFalpha,
TGF-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A, IL-17F, IL-18,
IL-19, IL-20, IL-23, IL-25, IFNalpha, MIF, ICAM-1, PGE4, PEG2, RANK
ligand, Te38, BAFF, CTLA-4, GP130, IL-12p40, VEGF, VEGF-A, PDGF,
HER1, HER2, HER3, HER3--BU, HER4, VEGF-C, VEGF-D, DR5, cMET, MET,
MET receptor, VEGFR, EGFR, CD40L, TNFR1, IL-1R, IL-6R, IL-18R,
EpCAM, MAPG, CSPGs, BTNO2, IGF1, IGF2, IGF1/2, Erb2B, MAG, NgR,
NogoA, NRP1, NRP2, OMGp, PDL-I, RGM A and RGM B. In some
embodiments, a heteromultimeric protein of this invention binds to
CD3 and at least one additional target molecule selected from BLR1,
BR3, CD19, CD20, CD22, CD72, CD79A, CD79B, CD180 (RP105), CR2,
FcRH1, FcRH2, FcRH5, FCER2, FCRL4, HLA-DOB, and NAG14.
First and second host cells in methods of the invention can be
cultured in any setting that permits expression and isolation of
the polypeptides of interest. For example, in one embodiment, the
first host cell and the second host cell in a method of the
invention are grown as separate cell cultures. In another
embodiment, the first host cell and the second host cell in a
method of the invention are grown as a mixed culture comprising
both host cells.
In some embodiments, at least one, at least two, at least three or
more additional hinge-containing polypeptide expressing host cells
may be grown either in the same or separate cultures as the first
and/or second hinge-containing host cells. In some embodiments, the
additional hinge-containing polypeptide(s) comprises the same
heterodimerization domain as the first hinge-containing
polypeptide. In some embodiments, the additional hinge-containing
polypeptide(s) comprises the same heterodimerization domain as the
second hinge-containing polypeptide.
Heteromultimeric proteins may be modified to enhance and/or add
additional desired characteristics. Such characteristics include
biological functions such as immune effector functions, a desirable
in vivo half life/clearance, bioavailability, biodistribution or
other pharmacokinetic characteristics. Such modifications are well
known in the art and can also be determined empirically, and may
include modifications by moieties that may or may not be
peptide-based. For example, antibodies may be glycosylated or
aglycosylated, generally depending at least in part on the nature
of the host cell. Preferably, antibodies of the invention are
aglycosylated. An aglycosylated antibody produced by a method of
the invention can subsequently be glycosylated by, for example,
using in vitro glycosylation methods well known in the art. As
described above and herein, heteromultimeric proteins of the
invention can be produced in a prokaryotic cell, such as, for
example, E. coli. E. coli-produced heteromultimeric proteins are
generally aglycosylated and lack the biological functions normally
associated with glycosylation profiles found in mammalian host cell
(e.g., CHO) produced heteromultimeric proteins.
The invention also provides immunoconjugates comprising a
heteromultimeric protein of the invention conjugated with a
heterologous moiety. Any heterologous moiety would be suitable so
long as its conjugation to the antibody does not substantially
reduce a desired function and/or characteristic of the antibody.
For example, in some embodiments, an immunoconjugate comprises a
heterologous moiety which is a cytotoxic agent. In some
embodiments, said cytotoxic agent is selected from the group
consisting of a radioactive isotope, a chemotherapeutic agent and a
toxin. In some embodiments, said toxin is selected from the group
consisting of calichemicin, maytansine and trichothene. In some
embodiments, an immunoconjugate comprises a heterologous moiety
which is a detectable marker. In some embodiments, said detectable
marker is selected from the group consisting of a radioactive
isotope, a member of a ligand-receptor pair, a member of an
enzyme-substrate pair and a member of a fluorescence resonance
energy transfer pair.
In one aspect, the invention provides compositions comprising a
heteromultimeric protein of the invention and a carrier, which in
some embodiments is pharmaceutically acceptable.
In another aspect, the invention provides compositions comprising
an immunoconjugate as described herein and a carrier, which in some
embodiments is pharmaceutically acceptable.
In one aspect, the invention provides a composition comprising a
population of multispecific heteromultimeric proteins of the
invention. As would be evident to one skilled in the art, generally
such a composition would not be completely (i.e., 100%)
homogeneous. However, as described herein, methods of the invention
are capable of producing a substantially homogeneous population of
multispecific heteromultimeric proteins. For example, the invention
provides a composition comprising heteromultimeric proteins,
wherein at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% of said heteromultimeric proteins are a
multispecific antibody (e.g., a bispecific antibody, etc.) of the
invention as described herein.
In one aspect, the invention provides a cell culture comprising a
mix of a first host cell and a second host cell, wherein the first
host cell comprises nucleic acid encoding a first hinge-containing
polypeptide, and the second host cell comprises nucleic acid
encoding a second hinge-containing polypeptide, and wherein the two
pairs have different target binding specificities. In one aspect,
the invention provides a cell culture comprising a mix of a first
host cell and a second host cell, wherein the first host cell
expresses a first pair of heavy and light chain polypeptides, and
the second host cell expresses a second pair of heavy and light
chain polypeptides, and wherein the two pairs have different target
binding specificities.
In another aspect, the invention provides articles of manufacture
comprising a container and a composition contained therein, wherein
the composition comprises a heteromultimeric protein (e.g., an
antibody) of the invention. In another aspect, the invention
provides articles of manufacture comprising a container and a
composition contained therein, wherein the composition comprises an
immunoconjugate as described herein. In some embodiments, these
articles of manufacture further comprise instructions for using
said composition.
In yet another aspect, the invention provides polynucleotides
encoding a heteromultimeric protein of the invention. In still
another aspect, the invention provides polynucleotides encoding an
immunoconjugate as described herein.
In one aspect, the invention provides recombinant vectors for
expressing a molecule (e.g., an antibody) of the invention. In
another aspect, the invention provides recombinant vectors for
expressing an immunoconjugate of the invention.
Any of a number of host cells can be used in methods of the
invention. Such cells are known in the art (some of which are
described herein) or can be determined empirically with respect to
suitability for use in methods of the invention using routine
techniques known in the art. In one embodiment, a host cell is
prokaryotic. In some embodiments, a host cell is a gram-negative
bacterial cell. In one embodiment, a host cell is E. coli. In some
embodiments, the E. coli is of a strain deficient in lipoprotein
(.DELTA.lpp). In some embodiments, the genotype of an E. coli host
cell lacks degP and prc genes and harbors a mutant spr gene. In one
embodiment, a host cell is mammalian, for example, a Chinese
Hamster Ovary (CHO) cell.
In one aspect, the invention provides host cells comprising a
polynucleotide or recombinant vector of the invention. In one
embodiment, a host cell is a mammalian cell, for example a Chinese
Hamster Ovary (CHO) cell. In one embodiment, a host cell is a
prokaryotic cell. In some embodiments, a host cell is a
gram-negative bacterial cell, which in some embodiments is E. coli.
Host cells of the invention may further comprise a polynucleotide
or recombinant vector encoding a molecule the expression of which
in a host cell enhances yield of a heteromultimeric protein in a
method of the invention. For example, such molecule can be a
chaperone protein. In one embodiment, said molecule is a
prokaryotic polypeptide selected from the group consisting of DsbA,
DsbC, DsbG and FkpA. In some embodiments, said polynucleotide or
recombinant vector encodes both DsbA and DsbC. In some embodiments,
an E. coli host cell is of a strain deficient in endogenous
protease activities. In some embodiments, the genotype of an E.
coli host cell is that of an E. coli strain that lacks degP and prc
genes and harbors a mutant spr gene. In some embodiments, the
genotype of an E. coli host cell is .DELTA.lpp.
Heteromultimeric proteins of the invention find a variety of uses
in a variety of settings. In one example, a heteromultimeric
protein of the invention is a therapeutic antibody. In another
example, a heteromultimeric protein of the invention is an agonist
antibody. In another example, a heteromultimeric protein of the
invention is an antagonistic antibody. A heteromultimeric protein
of the invention may also be a diagnostic antibody. In yet another
example, a heteromultimeric protein of the invention is a blocking
antibody. In another example, a heteromultimeric protein of the
invention is a neutralizing antibody.
In one aspect, the invention provides methods of treating or
delaying a disease in a subject, said methods comprising
administering a heteromultimeric protein of the invention to said
subject. In one embodiment, the disease is cancer. In another
embodiment, the disease is associated with dysregulation of
angiogenesis. In another embodiment, the disease is an immune
disorder, such as rheumatoid arthritis, immune thrombocytopenic
purpura, systemic lupus erythematosus, etc.
In one aspect, the invention provides use of a heteromultimeric
protein (e.g., an antibody) of the invention in the preparation of
a medicament for the therapeutic and/or prophylactic treatment of a
disease, such as a cancer, a tumor, a cell proliferative disorder,
an immune (such as autoimmune) disorder and/or an
angiogenesis-related disorder.
In one aspect, the invention provides use of a nucleic acid of the
invention in the preparation of a medicament for the therapeutic
and/or prophylactic treatment of a disease, such as a cancer, a
tumor, a cell proliferative disorder, an immune (such as
autoimmune) disorder and/or an angiogenesis-related disorder.
In one aspect, the invention provides use of an expression vector
of the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as a
cancer, a tumor, a cell proliferative disorder, an immune (such as
autoimmune) disorder and/or an angiogenesis-related disorder.
In one aspect, the invention provides use of a host cell of the
invention in the preparation of a medicament for the therapeutic
and/or prophylactic treatment of a disease, such as a cancer, a
tumor, a cell proliferative disorder, an immune (such as
autoimmune) disorder and/or an angiogenesis-related disorder.
In one aspect, the invention provides use of an article of
manufacture of the invention in the preparation of a medicament for
the therapeutic and/or prophylactic treatment of a disease, such as
a cancer, a tumor, a cell proliferative disorder, an immune (such
as autoimmune) disorder and/or an angiogenesis-related
disorder.
In one aspect, the invention provides use of a kit of the invention
in the preparation of a medicament for the therapeutic and/or
prophylactic treatment of a disease, such as a cancer, a tumor, a
cell proliferative disorder, an immune (such as autoimmune)
disorder and/or an angiogenesis-related disorder.
Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only, since various
changes and modifications within the scope and spirit of the
invention will become apparent to one skilled in the art from this
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates a fully oxidized half-antibody. Not shown are
the "knob" or "hole" or other heterodimerization domains. The
half-antibody depicted in this figure is an IgG1 isotype. One
skilled in the art will appreciate that the other immunoglobulin
isotypes can be envisioned as half-antibodies with the
corresponding inter- and intra-chain bonds. In an intact Ab the
hinge cysteines will form inter-chain disulfide bonds.
FIG. 1B illustrates a full-length bispecific antibody. Not depicted
are the inter-heavy chain disulfide bonds in the hinge region.
FIGS. 2A & B illustrates plasmids encoding the knob and hole
half-antibodies, respectively.
FIG. 3A illustrates the production of heteromultimeric proteins,
e.g., bispecific antibodies, using the common light chain method.
The produced BsAb has two different heavy chains with each being
paired with a common light chain.
FIG. 3B illustrates the production of heteromultimeric proteins,
e.g., bispecific antibodies, using separately engineered and
expressed half-antibodies. The produced BsAb typically has two
different heavy chains, each paired with its cognate light chain.
In this method each light chain is not necessarily the same for
each half-antibody.
FIG. 4A is a flow diagram for the production of bispecific
antibodies using separately engineered and expressed
half-antibodies. In this method, redox chemistry is used.
FIG. 4B shows a Coomassie stained gel. The two half-antibodies were
analyzed under reducing and non-reducing conditions by SDS-PAGE.
The predominant fraction is the 75 kD light chain-heavy chain pair
for each half-antibody under non-reducing conditions. Under
reducing conditions (e.g., treatment with DTT) each chain is
visible as a separate band.
FIG. 4C shows the results of ESI-TOF mass spectrometry of a
half-antibody with and without 1 mM N-ethylmaleimide (NEM)
treatment. No change in the mass of the half-antibody is observed
upon treatment with NEM indicating that all cysteines are fully
oxidized. The oxidized hinge cysteines are represented as a cyclic
disulfide in the depicted amino acid sequence. The expected mass
for the half-antibody is 72,548 Daltons, which is what is observed
by mass spectrometry indicating no covalent adducts.
FIG. 4D shows the carboxymethyl (CM) chromatogram, a photo of a
SDS-PAGE gel and the deconvoluted mass for the production of an
anti-EGFR/anti-c-met bispecific antibody. The CM chromatography
produces a single peak that is subsequently analyzed by SDS-PAGE.
The major band on the gel is the full-length (i.e., intact)
bispecific antibody. A minor band can also be seen at the 75 kD
range. The major band was subsequently analyzed by mass
spectrometry and indicated that the only detectable intact antibody
product was in agreement with theoretical MW of an
anti-EGFR/anti-c-met bispecific antibody.
FIG. 5A is a flow diagram for the large scale production of
bispecific antibodies using separately engineered and expressed
half-antibodies.
FIG. 5B is photograph of a gel showing the purified half-antibodies
were mostly the .about.75 kD species under non-reducing conditions.
Under reducing conditions (e.g., treatment with DTT) each chain is
visible as a separate band.
FIG. 5C shows the results of the SDS-PAGE analysis of the purified
bispecific after removal of aggregates indicating that the major
species is the intact bispecific antibody at 150 kD. Also shown are
the same samples under reducing conditions indicating that all
isolated product is either a light or heavy antibody chain.
FIG. 6A is a graph showing the biological activity of the
antibodies in a TF-2 cell proliferation assay testing
neutralization of the cytokines IL-4 and IL-13. The graph shows
that the bispecific possesses similar activity as the two
mammalian-produced, full-length antibodies added together or
separately.
FIG. 6B is a panel of three graphs showing the pharmacokinetic (PK)
properties of an anti-IL-4/anti-IL-13 bispecific antibody in
cynomologous monkey for the wild-type Fc and a mutated Fc as
determined by ELISA. The first graph shows the PK properties at a 2
mg/kg dose for the wild-type Fc. The middle graph shows the PK
properties at a 20 mg/kg dose, also for the wild-type Fc. The final
graph shows the PK properties at a 20 mg/kg dose for the mutant Fc.
The bispecific exhibits the expected two compartment clearances in
the animals tested. Females are represented by closed symbols and
males are represented by open symbols. In three animals, an
anti-therapeutic response was seen as indicated by the sharp
decrease in measured antibody in serum at day 21.
FIG. 7 is a photograph of a polyacrylamide gel. Whole fermentation
broth was mixed prior to lysis at varying ratios. After lysis
protein was extracted and loaded onto the gel under non-reducing
conditions. Purified bispecifics formed during this procedure are
visible as the top band on the gel.
FIG. 8A is a photograph of two polyacrylamide gels comparing the
bispecific antibody production when the cells are cultured
separately to a co-culture of the cells expressing the
half-antibodies. The intact bispecific forms to a much higher level
under co-culture conditions. When half-antibodies are expressed and
purified independently then mixed, the half-antibodies form less
than 5% of the intact bispecific. Under co-culture conditions,
greater than 40% is an intact bispecific as determined by 150
kD/(150 kD+75 kD) using L1-Core protein determinations.
FIG. 8B is a schematic of a co-culture experiment varying the cell
population of the initial inoculation. The ratios used and the
relative amount of full-length bispecific are shown at the bottom
of the figure.
FIG. 8C is a photograph of a gel for three separate 10 liter
fermentation runs of a 1:1 cell ratio of anti-EGFR and anti-c-Met.
Each run produced as the main product the full-length bispecific
indicating the reproducibility of the process.
FIG. 8D is a flow chart of the co-culture process for the
production of heteromultimeric proteins, e.g., bispecific
antibodies.
FIG. 8E is a chromatogram of the UV absorbance at 280 nm identified
two significant peaks at retention times 91.79 and 102.35.
Subsequent analysis by mass spectrometry indicated that the intact
bispecific antibody was effectively separated from the excess
half-antibody.
FIG. 8F shows the analysis of Peak 91.79 from FIG. 8E by SDS-PAGE
and mass spectrometry. Decovolution of mass spectrometry data
produced a single peak at 146,051.89 Daltons, which is in agreement
with the expected mass of the bispecific antibody. Contaminating
homodimeric species were not detected.
FIG. 8G is a comparison of the work flows for independent
production and co-culture production of heteromultimeric
proteins.
FIG. 9A show three chromatograms. The top chromatogram shows no
absorbance peak during the elution for the sample without EDTA. The
middle chromatogram shows the sample with EDTA has a distinct
elution peak from which we recovered approximately 1.5 mg protein.
The lower chromatogram shows the sample treated with EDTA and Mg
also showed a similar elution peak from which we recovered 1.1 mg
protein. Recovered proteins from the EDTA sample, EDTA plus Mg
sample, and a pool of fractions from the same retention time from
the untreated EDTA sample were analyzed by SDS-PAGE under reducing
and non-reducing conditions.
FIG. 9B is a photograph of the SDS-PAGE gel described in FIG. 9A.
The samples treated with EDTA have produced intact bispecific
antibody that has been released into the culture media.
FIG. 9C-1, FIG. 9C-2 and FIG. 9C-3 show the mass spec chromatograms
for the samples recovered and described in FIG. 9A. The samples
with the EDTA showed the expected mass for the bispecific antibody
and a mass for the excess half-antibody.
FIG. 9D is a photograph of a SDS-PAGE gel and mass chromatograms of
the indicated bands. Lane is MW markers, Lane 2 is anti-IL-13
independently expressed, Lane 3 is antiI-IL-4 independently
expressed and Lane 4 is a co-culture of the two cells. Mass spec
analysis of all three samples shows that the co-culture produces
the intact bispecific and an excess of one half-antibody,
anti-IL-4. This indicates the anti-IL-13 half-antibody is
stoichiometrically limiting. When half-antibodies are expressed and
purified independently then mixed, the half-antibodies form
approximately 2% (anti-IL-13) and 3% (anti-IL-4) of the intact
bispecific. Under co-culture conditions, approximately 60% is an
intact bispecific as determined by 150 kD/(150 kD+75 kD) using
L1-Core protein determinations.
FIG. 9E-1 and FIG. 9E-2 show two HIC chromatograms for two
co-cultures that had different cell ratios in the initial
fermentation inoculum as indicated. A clear difference in the
product is observed that reflects the initial inoculum ratio. Using
this approach it becomes apparent that the initial inoculum ratio
can be altered to achieve optimum production of the
heteromultimeric protein.
FIG. 9F is a panel of four photographs showing the SDS-PAGE
analysis under reducing and non-reducing conditions of eight
different bispecific antibodies produced by the co-culture process
described herein The non-reducing gels for the anti-CD3/anti-CD19
heteromultimeric proteins is not shown. Arrows indicate the intact
bispecific antibodies.
FIG. 10 is a schematic of a matrix approach to screening
heteromultimeric proteins.
FIG. 11 shows two graphs for in vitro activity of bispecific
antibodies produced using the methods described herein.
FIG. 12 is a graph showing that the anti-EGFR/anti-c-met bispecific
possesses anti-tumor activity in a KP4 pancreatic xenograft in vivo
model.
FIG. 13 is a graph showing that the anti-EGFR/anti-c-met bispecific
possesses anti-tumor activity in an A431 epidermoid carcinoma
xenograft in vivo model.
FIG. 14 shows the HIC of A) knob pre-assembly B) hole pre-assembly
C) bispecific post assembly. FIG. 14D is a gel of each arm
pre-assembly
FIG. 15 shows an electrophoretogram of assembled material
indicating that 86% of the material is fully oxidized.
FIG. 16: Characterization of assembled bispecific A) HIC
chromatogram of annealed bispecific indicates that the material is
>90.5 percent bispecific B) gel of purified material C) mass
spectronomy deconvolution of final sample, and D) table of
theoretical masses.
FIG. 17 is a schematic of redox procedure (with heat): a) sample is
heated for an hour to allow cyclisation of disulfide bonds, b) then
cooled and cysteines are reduced using 2 mM DTT for two hours, and
c) then concentrated and cysteines are air oxidized by dialysis at
room temperature.
FIG. 18 is a schematic of redox procedure (without heat): a) sample
is mixed for two hours, b) cysteines are reduced using 2 mM DTT for
two hours, and c) then concentrated and cysteines are air oxidized
while EDTA is removed by dialysis at room temperature
FIG. 19: Analytics of assembled bispecific A) HIC chromatogram
using redox procedure with heating step B) HIC chromatogram using
redox procedure without heating step.
ABBREVIATIONS
ADCC=Antibody-dependent cell-mediated cytotoxicity
API=Anti-pathogen immunoadhesins
BPI=Bactericidal/permeability-increasing protein
C1q=Complement factor 1q
CD=Cluster of Differentiation
CDC=Complement-dependent cytotoxicity
CH1 or C.sub.H1=Heavy chain first constant domain
CH2 or C.sub.H2=Heavy chain second constant domain
CH3 or C.sub.H3=Heavy chain third constant domain
CH4 or C.sub.H4=Heavy chain fourth constant domain
CL or C.sub.L=Light chain constant domain
CTLA=Cytotoxic T lymphocyte-associated molecule
Fc=Fragment crystallizable
Fc.gamma.R=Receptor gamma for the Fc portion of IgG
HIV=Human immunodeficiency virus
ICAM=Intercellular adhesion molecule
BsAb=Bispecific antibody
BsDb=Bispecific diabody
dsFv=Disulfide-stabilized Fv
Fc=Constant fragment of an antibody
Fd=V.sub.H+C.sub.H1 of an antibody
FcR=Fc receptor
Fv=Variable fragment of an antibody
IgG=Immunoglobulin G
mAb=Monoclonal antibody
PBL=Peripheral blood lymphocyte
scDb=Single-chain diabody
scFv=Single-chain Fv
(scFv).sub.2=scFv-scFv tandem
Tandab=Tandem diabody
VH or V.sub.H=Variable domain of the heavy chain of an antibody
VL or V.sub.L=Variable domain of the light chain of an antibody
DETAILED DESCRIPTION
The invention will now be described in detail by way of reference
only using the following definitions and examples. All patents and
publications, including all sequences disclosed within such patents
and publications, referred to herein are expressly incorporated by
reference.
Unless defined otherwise herein, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with a general
dictionary of many of the terms used in this invention. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
Numeric ranges are inclusive of the numbers defining the range.
Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively. Practitioners
are particularly directed to Sambrook et al., 1989, and Ausubel F M
et al., 1993, for definitions and terms of the art. It is to be
understood that this invention is not limited to the particular
methodology, protocols, and reagents described, as these may
vary.
Numeric ranges are inclusive of the numbers defining the range.
Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively.
The headings provided herein are not limitations of the various
aspects or embodiments of the invention which can be had by
reference to the specification as a whole. Accordingly, the terms
defined immediately below are more fully defined by reference to
the specification as a whole.
I. DEFINITIONS
A "heteromultimer", "heteromultimeric complex", or
"heteromultimeric protein" refers to a molecule comprising at least
a first hinge-containing polypeptide and a second hinge-containing
polypeptide, wherein the second hinge-containing polypeptide
differs in amino acid sequence from the first hinge-containing
polypeptide by at least one amino acid residue. The heteromultimer
can comprise a "heterodimer" formed by the first and second
hinge-containing polypeptides or can form higher order tertiary
structures where polypeptides in addition to the first and second
hinge-containing polypeptides are present. The polypeptides of the
heteromultimer may interact with each other by a non-peptidic,
covalent bond (e.g., disulfide bond) and/or a non-covalent
interaction (e.g., hydrogen bonds, ionic bonds, van der Waals
forces, and/or hydrophobic interactions).
As used herein, "heteromultimerization domain" refers to
alterations or additions to a biological molecule so as to promote
heteromultimer formation and hinder homomultimer formation. Any
heterodimerization domain having a strong preference for forming
heterodimers over homodimers is within the scope of the invention.
Illustrative examples include but are not limited to, for example,
US Patent Application 20030078385 (Arathoon et al.--Genentech;
describing knob into holes); WO2007147901 (Kj.ae butted.rgaard et
al.--Novo Nordisk: describing ionic interactions); WO 2009089004
(Kannan et al.--Amgen: describing electrostatic steering effects);
U.S. Provisional Patent Application 61/243,105 (Christensen et
al.--Genentech; describing coiled coils). See also, for example,
Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992)
describing leucine zipper or Pack et al., Bio/Technology 11,
1271-1277 (1993) describing the helix-turn-helix motif. The phrase
"heteromultimerization domain" and "heterodimerization domain" are
used interchangeably herein.
The phrase "hinge-containing polypeptide" as used herein refers to
a polypeptide that comprises a region corresponding to the hinge
region of an immunoglobulin as understood in the art, e.g., the
region between the C.sub.H1 and C.sub.H2 domains of the heavy
chain. The "hinge region," "hinge sequence", and variations
thereof, as used herein, includes the meaning known in the art,
which is illustrated in, for example, Janeway's Immunobiology,
(Garland Science, Taylor & Francis Group, LLC, NY) (7th ed.,
2008); Bloom et al., Protein Science (1997), 6:407-415; Humphreys
et al., J. Immunol. Methods (1997), 209:193-202. See also, for
example, Burton, Molec. Immunol. 22:161-206 (1985) and Papadea, C.
and I. J. Check (1989) "Human immunoglobulin G and immunoglobulin G
subclasses: biochemical, genetic, and clinical aspects." Crit. Rev
Clin Lab Sci 27(1): 27-58. It will be appreciated by one skilled in
the art that the number of amino acids as well as the number of
cysteine residues available for interchain disulfide bond formation
varies between the classes and isotypes of immunoglobulins. All
such hinge regions may be in the hinge-containing polypeptides and
are within the scope of the invention.
The term "antibody" herein is used in the broadest sense and refers
to any immunoglobulin (Ig) molecule comprising two heavy chains and
two light chains, and any fragment, mutant, variant or derivation
thereof so long as they exhibit the desired biological activity
(e.g., epitope binding activity). Examples of antibodies include
monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies) and antibody fragments as
described herein. An antibody can be human, humanized and/or
affinity matured.
As a frame of reference, as used herein an antibody will refer to
the structure of an immunoglobulin G (IgG). However, one skilled in
the art would understand/recognize that an antibody of any
immunoglobulin class may be utilized in the inventive method
described herein. For clarity, an IgG molecule contains a pair of
identical heavy chains (HCs) and a pair of identical light chains
(LCs). Each LC has one variable domain (V.sub.L) and one constant
domain (C.sub.L), while each HC has one variable (V.sub.H) and
three constant domains (C.sub.H1, C.sub.H2, and C.sub.H3). The
C.sub.H1 and C.sub.H2 domains are connected by a hinge region. This
structure is well known in the art. Reference is made to FIG.
1B.
As used herein, "half-antibody" refers to one immunoglobulin heavy
chain associated with one immunoglobulin light chain. An exemplary
half-antibody is depicted in FIG. 1A. One skilled in the art will
readily appreciate that a half-antibody may also have an antigen
binding domain consisting of a single variable domain.
The term "maxibody" refers to a fusion protein comprising a scFv
fused to an Fc polypeptide. Reference is made to FIG. 8a of WO
2009089004. Reference is made to FIG. 2 of WO 2009089004 for a
bispecific maxibody.
The term "C.sub.H2 domain" of a human IgG Fc region usually extends
from about residues 231 to about 340 of the IgG according to the EU
numbering system. The C.sub.H2 domain is unique in that it is not
closely paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two C.sub.H2 domains
of an intact native IgG molecule. It has been speculated that the
carbohydrate may provide a substitute for the domain-domain pairing
and help stabilize the C.sub.H2 domain. Burton, Molec. Immunol.
22:161-206 (1985).
The term "C.sub.H3 domain" comprises the stretch of residues
C-terminal to a C.sub.H2 domain in an Fc region (i.e., from about
amino acid residue 341 to about amino acid residue 447 of an IgG
according to the EU numbering system).
The term "Fc region", as used herein, generally refers to a dimer
complex comprising the C-terminal polypeptide sequences of an
immunoglobulin heavy chain, wherein a C-terminal polypeptide
sequence is that which is obtainable by papain digestion of an
intact antibody. The Fc region may comprise native or variant Fc
sequences. Although the boundaries of the Fc sequence of an
immunoglobulin heavy chain might vary, the human IgG heavy chain Fc
sequence is usually defined to stretch from an amino acid residue
at about position Cys226, or from about position Pro230, to the
carboxyl terminus of the Fc sequence. Unless otherwise specified
herein, numbering of amino acid residues in the Fc region or
constant region is according to the EU numbering system, also
called the EU index, as described in Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md., 1991. The Fc sequence
of an immunoglobulin generally comprises two constant domains, a
C.sub.H2 domain and a C.sub.H3 domain, and optionally comprises a
C.sub.H4 domain. By "Fc polypeptide" herein is meant one of the
polypeptides that make up an Fc region, e.g., a monomeric Fc. An Fc
polypeptide may be obtained from any suitable immunoglobulin, such
as IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4 subtypes, IgA,
IgE, IgD or IgM. The Fc region comprises the carboxy-terminal
portions of both H chains held together by disulfides. The effector
functions of antibodies are determined by sequences in the Fc
region; this region is also the part recognized by Fc receptors
(FcR) found on certain types of cells. In some embodiments, an Fc
polypeptide comprises part or all of a wild type hinge sequence
(generally at its N terminus). In some embodiments, an Fc
polypeptide does not comprise a functional or wild type hinge
sequence.
A "functional Fc region" possesses an "effector function" of a
native sequence Fc region. Exemplary "effector functions" include
C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down
regulation of cell surface receptors (e.g., B cell receptor; BCR),
etc. Such effector functions generally require the Fc region to be
combined with a binding domain (e.g., an antibody variable domain)
and can be assessed using various assays as disclosed, for example,
in definitions herein.
A "native sequence Fc region" comprises an amino acid sequence
identical to the amino acid sequence of an Fc region found in
nature. Native sequence human Fc regions include a native sequence
human IgG.sub.1 Fc region (non-A and A allotypes); native sequence
human IgG.sub.2 Fc region; native sequence human IgG.sub.3 Fc
region; and native sequence human IgG.sub.4 Fc region as well as
naturally occurring variants thereof.
A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g., from
about one to about ten amino acid substitutions, and preferably
from about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95%, at
least about 96%, at least about 97%, at least about 98% or at least
about 99% homology therewith.
"Fc component" as used herein refers to a hinge region, a C.sub.H2
domain or a C.sub.H3 domain of an Fc region.
In certain embodiments, the hinge-containing polypeptide comprises
an IgG Fc region, preferably derived from a wild-type human IgG Fc
region. By "wild-type" human IgG Fc it is meant a sequence of amino
acids that occurs naturally within the human population. Of course,
just as the Fc sequence may vary slightly between individuals, one
or more alterations may be made to a wildtype sequence and still
remain within the scope of the invention. For example, the Fc
region may contain additional alterations that are not related to
the present invention, such as a mutation in a glycosylation site
or inclusion of an unnatural amino acid.
The term "variable region" or "variable domain" refers to the
domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (V.sub.H and V.sub.L, respectively) of a
native antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single V.sub.H or V.sub.L domain may be sufficient to confer
antigen-binding specificity. Furthermore, antibodies that bind a
particular antigen may be isolated using a V.sub.H or V.sub.L
domain from an antibody that binds the antigen to screen a library
of complementary V.sub.L or V.sub.H domains, respectively. See,
e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et
al., Nature 352:624-628 (1991).
The term "Fab" as used herein refers to an antigen-binding fragment
of an antibody. As noted above, papain may be used to digest an
intact antibody. Papain digestion of antibodies produces two
identical antigen-binding fragments, i.e., "Fab" fragments, and a
residual "Fc" fragment (i.e., the Fc region, supra). The Fab
fragment consists of an entire L chain along with the variable
region domain of the H chain (V.sub.H), and the first constant
domain of one heavy chain (C.sub.H1).
The phrase "antigen binding arm", "target molecule binding arm",
"target binding arm" and variations thereof, as used herein, refers
to a component part of a heteromultimeric protein of the invention
that has an ability to specifically bind a target of interest.
Generally and preferably, the antigen binding arm is a complex of
immunoglobulin polypeptide sequences, e.g., CDR and/or variable
domain sequences of an immunoglobulin light and heavy chain.
A "target" or "target molecule" refers to a moiety recognized by a
binding arm of the heteromultimeric protein. For example, if the
heteromultimeric protein is an antibody, then the target may be
epitopes on a single molecule or on different molecules, or a
pathogen or a tumor cell, depending on the context. Similarly, if
the heteromultimeric protein is a receptor-Fc fusion protein the
target would be the cognate binding partner for the receptor. One
skilled in the art will appreciate that the target is determined by
the binding specificity of the target binding arm and that
different target binding arms may recognize different targets. A
target preferably binds to a heteromultimeric protein of this
invention with affinity higher than 1 uM Kd (according to scatchard
analysis). Examples of target molecules include, but are not
limited to, serum soluble proteins and/or their receptors, such as
cytokines and/or cytokine receptors, adhesins, growth factors
and/or their receptors, hormones, viral particles (e.g., RSV F
protein, CMV, StaphA, influenza, hepatitis C virus), micoorganisms
(e.g., bacterial cell proteins, fungal cells), adhesins, CD
proteins and their receptors.
One example of an "intact" or "full-length" antibody is one that
comprises an antigen-binding arm as well as a C.sub.L and at least
heavy chain constant domains, C.sub.H1, C.sub.H2, and C.sub.H3. The
constant domains can be native sequence constant domains (e.g.,
human native sequence constant domains) or amino acid sequence
variants thereof.
The term "coupling" as used herein refers to the steps necessary to
link the first and second hinge-containing polypeptides to each
other, e.g., formation of a covalent bond. Such steps comprise the
reducing, annealing and/or oxidizing of cysteine residues in the
first and second hinge-containing polypeptides to form an
inter-chain disulfide bond. The coupling may be achieved by
chemical cross-linking or the use of a redox system. See, e.g.,
Humphreys et al., J. Immunol. Methods (1998) 217:1-10 and Zhu et
al., Cancer Lett., (1994) 86: 127-134.
The term "multispecific antibody" is used in the broadest sense and
specifically covers an antibody that has polyepitopic specificity.
Such multispecific antibodies include, but are not limited to, an
antibody comprising a heavy chain variable domain (V.sub.H) and a
light chain variable domain (V.sub.L), wherein the V.sub.HV.sub.L
unit has polyepitopic specificity, antibodies having two or more
V.sub.L and V.sub.H domains with each V.sub.HV.sub.L unit binding
to a different epitope, antibodies having two or more single
variable domains with each single variable domain binding to a
different epitope, full length antibodies, antibody fragments such
as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and
triabodies, antibody fragments that have been linked covalently or
non-covalently. "Polyepitopic specificity" refers to the ability to
specifically bind to two or more different epitopes on the same or
different target(s). "Monospecific" refers to the ability to bind
only one epitope. According to one embodiment the multispecific
antibody is an IgG antibody that binds to each epitope with an
affinity of 5 .mu.M to 0.001 pM, 3 .mu.M to 0.001 pM, 1 .mu.M to
0.001 pM, 0.5 .mu.M to 0.001 pM, or 0.1 .mu.M to 0.001 pM. An
illustrative drawing of a bispecific is provided in FIG. 1B.
"Antibody fragments" comprise a portion of an intact antibody,
preferably the antigen binding or a variable region of the intact
antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies (Db); tandem diabodies
(taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870; Zapata et
al., Protein Eng. 8(10):1057-1062 (1995)); one-armed antibodies,
single variable domain antibodies, minibodies, single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments (e.g., including but not limited to, Db-Fc,
taDb-Fc, taDb-C.sub.H3 and (scFV)4-Fc).
The expression "single domain antibodies" (sdAbs) or "single
variable domain (SVD) antibodies" generally refers to antibodies in
which a single variable domain (V.sub.H or V.sub.L) can confer
antigen binding. In other words, the single variable domain does
not need to interact with another variable domain in order to
recognize the target antigen. Single domain antibodies consist of a
single monomeric variable antibody domain (V.sub.H or V.sub.L) on
each antigen binding arm. Examples of single domain antibodies
include those derived from camelids (llamas and camels) and
cartilaginous fish (e.g., nurse sharks) and those derived from
recombinant methods from humans and mouse antibodies (Ward et al.,
Nature (1989) 341:544-546; Dooley and Flajnik, Dev Comp Immunol
(2006) 30:43-56; Muyldermans et al., Trend Biochem Sci (2001)
26:230-235; Holt et al., Trends Biotechnol (2003):21:484-490; WO
2005/035572; WO 03/035694; Davies and Riechmann, Febs Lett (1994)
339:285-290; WO00/29004; WO 02/051870). A single variable domain
antibody can be present in an antigen binding arm (e.g., homo- or
hetero-multimer) with other variable regions or variable domains,
in which case it is not a single domain antibody.
The expression "linear antibodies" generally refers to the
antibodies described in Zapata et al., Protein Eng. 8(10):1057-1062
(1995). Briefly, these antibodies comprise a pair of tandem Fd
segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
The term "knob-into-hole" or "KnH" technology as mentioned herein
refers to the technology directing the pairing of two polypeptides
together in vitro or in vivo by introducing a protuberance (knob)
into one polypeptide and a cavity (hole) into the other polypeptide
at an interface in which they interact. For example, KnHs have been
introduced in the Fc:Fc binding interfaces, C.sub.L:C.sub.H1
interfaces or V.sub.H/V.sub.L interfaces of antibodies (e.g.,
US2007/0178552, WO 96/027011, WO 98/050431 and Zhu et al. (1997)
Protein Science 6:781-788). This is especially useful in driving
the pairing of two different heavy chains together during the
manufacture of multispecific antibodies. For example, multispecific
antibodies having KnH in their Fc regions can further comprise
single variable domains linked to each Fc region, or further
comprise different heavy chain variable domains that pair with
similar or different light chain variable domains. KnH technology
can be also be used to pair two different receptor extracellular
domains together or any other polypeptide sequences that comprises
different target recognition sequences (e.g., including affibodies,
peptibodies and other Fc fusions).
"Fv" consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although often at a lower affinity
than the entire binding site.
"Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody
fragments that comprise the V.sub.H and V.sub.L antibody domains
connected into a single polypeptide chain. Preferably, the sFv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains, which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Malmborg et al., J. Immunol. Methods 183:7-13, 1995.
The term "diabodies" refers to small antibody fragments prepared by
constructing sFv fragments (see preceding paragraph) with short
linkers (about 5-10 residues) between the V.sub.H and V.sub.L
domains such that inter-chain but not intra-chain pairing of the V
domains is achieved, resulting in a bivalent fragment, i.e.,
fragment having two antigen-binding sites. Bispecific diabodies are
heterodimers of two "crossover" sFv fragments in which the V.sub.H
and V.sub.L domains of the two antibodies are present on different
polypeptide chains. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993).
The term "one-armed antibody" or "one-armed antibodies" refers to
an antibody that comprises (1) a variable domain joined by a
peptide bond to polypeptide comprising a C.sub.H2 domain, a
C.sub.H3 domain or a C.sub.H2-C.sub.H3 domain and (2) a second
C.sub.H2, C.sub.H3 or C.sub.H2-C.sub.H3 domain, wherein a variable
domain is not joined by a peptide bond to a polypeptide comprising
the second C.sub.H2, C.sub.H3 or C.sub.H2-C.sub.H3 domain. In one
embodiment, the one-armed antibody comprises 3 polypeptides (1) a
first polypeptide comprising a variable domain (e.g., V.sub.H),
C.sub.H1, C.sub.H2 and C.sub.H3, (2) a second polypeptide
comprising a variable domain (e.g., V.sub.L) and a C.sub.L domain,
and (3) a third polypeptide comprising a C.sub.H2 and C.sub.H3
domain. In another embodiment, the one-armed antibody has a partial
hinge region containing the two cysteine residues which form
disulphide bonds linking the constant heavy chains. In one
embodiment, the variable domains of the one armed antibody form an
antigen binding region. In another embodiment, the variable domains
of the one armed antibody are single variable domains, wherein each
single variable domain is an antigen binding region. In an
embodiment, the one-armed antibody is a single variable domain
antibody.
Antibodies of the invention can be "chimeric" antibodies in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, provided that they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; and
Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
Chimeric antibodies of interest herein include primatized
antibodies comprising variable domain antigen-binding sequences
derived from a non-human primate (e.g., Old World Monkey, Ape,
etc.) and human constant region sequences.
"Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
"Peptibody" or "peptibodies" refers to a fusion of randomly
generated peptides with an Fc domain. See U.S. Pat. No. 6,660,843,
issued Dec. 9, 2003 to Feige et al. (incorporated by reference in
its entirety). They include one or more peptides linked to the
N-terminus, C-terminus, amino acid sidechains, or to more than one
of these sites. Peptibody technology enables design of therapeutic
agents that incorporate peptides that target one or more ligands or
receptors, tumor-homing peptides, membrane-transporting peptides,
and the like. Peptibody technology has proven useful in design of a
number of such molecules, including linear and
disulfide-constrained peptides, "tandem peptide multimers" (i.e.,
more than one peptide on a single chain of an Fc domain). See, for
example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156,
published Oct. 16, 2003 (corresponding to WO 02/092620, published
Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18,
2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S.
Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO
00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023,
published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003;
U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003
(corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser.
No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329,
published Apr. 1, 2004), each of which is hereby incorporated by
reference in its entirety.
"Affibodies" or "Affibody" refers to the use of a protein liked by
peptide bond to an Fc region, wherein the protein is used as a
scaffold to provide a binding surface for a target molecule. The
protein is often a naturally occurring protein such as
staphylococcal protein A or IgG-binding B domain, or the Z protein
derived therefrom (see Nilsson et al (1987), Prot Eng 1, 107-133,
and U.S. Pat. No. 5,143,844) or a fragment or derivative thereof.
For example, affibodies can be created from Z proteins variants
having altered binding affinity to target molecule(s), wherein a
segment of the Z protein has been mutated by random mutagenesis to
create a library of variants capable of binding a target molecule.
Examples of affibodies include U.S. Pat. No. 6,534,628, Nord K et
al, Prot Eng 8:601-608 (1995) and Nord K et al, Nat Biotech
15:772-777 (1997). Biotechnol Appl Biochem. 2008 June; 50(Pt
2):97-112.
As used herein, the term "immunoadhesin" designates molecules which
combine the binding specificity of a heterologous protein (an
"adhesin") with the effector functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of an
amino acid sequence with a desired binding specificity, which amino
acid sequence is other than the antigen recognition and binding
site of an antibody (i.e., is "heterologous" compared to a constant
region of an antibody), and an immunoglobulin constant domain
sequence (e.g., C.sub.H2 and/or C.sub.H3 sequence of an IgG).
Exemplary adhesin sequences include contiguous amino acid sequences
that comprise a portion of a receptor or a ligand that binds to a
protein of interest. Adhesin sequences can also be sequences that
bind a protein of interest, but are not receptor or ligand
sequences (e.g., adhesin sequences in peptibodies). Such
polypeptide sequences can be selected or identified by various
methods, include phage display techniques and high throughput
sorting methods. The immunoglobulin constant domain sequence in the
immunoadhesin can be obtained from any immunoglobulin, such as
IgG1, IgG2, IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2),
IgE, IgD, or IgM.
"Complex" or "complexed" as used herein refers to the association
of two or more molecules that interact with each other through
bonds and/or forces (e.g., van der waals, hydrophobic, hydrophilic
forces) that are not peptide bonds. In one embodiment, the complex
is heteromultimeric. It should be understood that the term "protein
complex" or "polypeptide complex" as used herein includes complexes
that have a non-protein entity conjugated to a protein in the
protein complex (e.g., including, but not limited to, chemical
molecules such as a toxin or a detection agent).
A heteromultimeric protein of this invention "which binds an
antigen of interest is one that binds the target with sufficient
affinity such that the heteromultimeric protein is useful as a
diagnostic and/or therapeutic agent in targeting a protein or a
cell or tissue expressing the target, and does not significantly
cross-react with other proteins. In such embodiments, the extent of
binding of the heteromultimeric protein to a "non-target" protein
will be less than about 10% of the binding of the antibody to its
particular target protein as determined by fluorescence activated
cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) or
ELISA. With regard to the binding of a heteromultimeric protein to
a target molecule, the term "specific binding" or "specifically
binds to" or is "specific for" a particular polypeptide or an
epitope on a particular polypeptide target means binding that is
measurably different from a non-specific interaction (e.g., a
non-specific interaction may be binding to bovine serum albumin or
casein). Specific binding can be measured, for example, by
determining binding of a molecule compared to binding of a control
molecule. For example, specific binding can be determined by
competition with a control molecule that is similar to the target,
for example, an excess of non-labeled target. In this case,
specific binding is indicated if the binding of the labeled target
to a probe is competitively inhibited by excess unlabeled target.
The term "specific binding" or "specifically binds to" or is
"specific for" a particular polypeptide or an epitope on a
particular polypeptide target as used herein can be exhibited, for
example, by a molecule having a Kd for the target of at least about
200 nM, alternatively at least about 150 nM, alternatively at least
about 100 nM, alternatively at least about 60 nM, alternatively at
least about 50 nM, alternatively at least about 40 nM,
alternatively at least about 30 nM, alternatively at least about 20
nM, alternatively at least about 10 nM, alternatively at least
about 8 nM, alternatively at least about 6 nM, alternatively at
least about 4 nM, alternatively at least about 2 nM, alternatively
at least about 1 nM, or greater. In one embodiment, the term
"specific binding" refers to binding where a heteromultimeric
protein binds to a particular polypeptide or epitope on a
particular polypeptide without substantially binding to any other
polypeptide or polypeptide epitope.
"Binding affinity" generally refers to the strength of the sum
total of noncovalent interactions between a single binding site of
a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd). For
example, the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM,
40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or
stronger. Affinity can be measured by common methods known in the
art, including those described herein. Low-affinity antibodies
generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and
tend to remain bound longer. A variety of methods of measuring
binding affinity are known in the art, any of which can be used for
purposes of the present invention.
In one embodiment, the "Kd" or "Kd value according to this
invention is measured by using surface plasmon resonance assays
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized target (e.g.,
antigen) CM5 chips at .about.10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium
acetate, pH 4.8, into 5 .mu.g/ml (.about.0.2 .mu.M) before
injection at a flow rate of 5 .mu.l/minute to achieve approximately
10 response units (RU) of coupled protein. Following the injection
of antigen, 1 M ethanolamine is injected to block unreacted groups.
For kinetics measurements, two-fold serial dilutions of Fab (e.g.,
0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST)
at 25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (k.sub.on) and dissociation rates (k.sub.off) are
calculated using a simple one-to-one Langmuir binding model
(BIAcore Evaluation Software version 3.2) by simultaneous fitting
the association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999). If the on-rate exceeds 10.sup.6 M.sup.-1 the
surface plasmon resonance assay above, then the on-rate can be
determined by using a fluorescent quenching technique that measures
the increase or decrease in fluorescence emission intensity
(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25.degree.
C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in
the presence of increasing concentrations of antigen as measured in
a spectrometer, such as a stop-flow equipped spectrophotometer
(Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer
(ThermoSpectronic) with a stirred cuvette.
"Biologically active" and "biological activity" and "biological
characteristics" with respect to a heteromultimeric protein of this
invention, such as an antibody, fragment, or derivative thereof,
means having the ability to bind to a biological molecule, except
where specified otherwise.
"Isolated," when used to describe the various heteromultimer
polypeptides means a heteromultimer which has been separated and/or
recovered from a cell or cell culture from which it was expressed.
Contaminant components of its natural environment are materials
which would interfere with diagnostic or therapeutic uses for the
heteromultimer, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In certain embodiments,
the heteromultimer will be purified (1) to greater than 95% by
weight of protein as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Ordinarily, however,
isolated polypeptide will be prepared by at least one purification
step.
The heteromultimers of the present invention are generally purified
to substantial homogeneity. The phrases "substantially
homogeneous", "substantially homogeneous form" and "substantial
homogeneity" are used to indicate that the product is substantially
devoid of by-products originated from undesired polypeptide
combinations (e.g., homomultimers).
Expressed in terms of purity, substantial homogeneity means that
the amount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%,
3%, 2% or 1% by weight or is less than 1% by weight. In one
embodiment, the by-product is below 5%.
"Biological molecule" refers to a nucleic acid, a protein, a
carbohydrate, a lipid, and combinations thereof. In one embodiment,
the biologic molecule exists in nature.
By "linked" or "links as used herein is meant either a direct
peptide bond linkage between a first and second amino acid sequence
or a linkage that involves a third amino acid sequence that is
peptide bonded to and between the first and second amino acid
sequences. For example, a linker peptide bonded to the C-terminal
end of one amino acid sequence and to the N-terminal end of the
other amino acid sequence.
By "linker" as used herein is meant an amino acid sequence of two
or more amino acids in length. The linker can consist of neutral
polar or nonpolar amino acids. A linker can be, for example, 2 to
100 amino acids in length, such as between 2 and 50 amino acids in
length, for example, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50
amino acids in length. A linker can be "cleavable," for example, by
auto-cleavage, or enzymatic or chemical cleavage. Cleavage sites in
amino acid sequences and enzymes and chemicals that cleave at such
sites are well known in the art and are also described herein.
By a "tether" as used herein is meant an amino acid linker that
joins two other amino acid sequences. A tether as described herein
can link the N-terminus of an immunoglobulin heavy chain variable
domain with the C-terminus of an immunoglobulin light chain
constant domain. In particular embodiments, a tether is between
about 15 and 50 amino acids in length, for example, between 20 and
26 amino acids in length (e.g., 20, 21, 22, 23, 24, 25, or 26 amino
acids in length). A tether may be "cleavable," for example, by
auto-cleavage, or enzymatic or chemical cleavage using methods and
reagents standard in the art.
Enzymatic cleavage of a "linker" or a "tether" may involve the use
of an endopeptidase such as, for example, Lys-C, Asp-N, Arg-C, V8,
Glu-C, chymotrypsin, trypsin, pepsin, papain, thrombin, Genenase,
Factor Xa, TEV (tobacco etch virus cysteine protease),
Enterokinase, HRV C3 (human rhinovirus C3 protease), Kininogenase,
as well as subtilisin-like proprotein convertases (e.g., Furin
(PC1), PC2, or PC3) or N-arginine dibasic convertase. Chemical
cleavage may involve use of, for example, hydroxylamine,
N-chlorosuccinimide, N-bromosuccinimide, or cyanogen bromide.
A "Lys-C endopeptidase cleavage site" as used herein is a Lysine
residue in an amino acid sequence that can be cleaved at the
C-terminal side by Lys-C endopeptidase. Lys-C endopeptidase cleaves
at the C-terminal side of a Lysine residue.
By a "chaotropic agent" is meant a water-soluble substance which
disrupts the three-dimensional structure of a protein (e.g., an
antibody) by interfering with stabilizing intra-molecular
interactions (e.g., hydrogen bonds, van der Waals forces, or
hydrophobic effects). Exemplary chaotropic agents include, but are
not limited to, urea, Guanidine-HCl, lithium perchlorate,
Histidine, and Arginine.
By a "mild detergent" is meant a water-soluble substance which
disrupts the three-dimensional structure of a protein (e.g., an
antibody) by interfering with stabilizing intra-molecular
interactions (e.g., hydrogen bonds, van der Waals forces, or
hydrophobic effects), but which does not permanently disrupt the
protein structure as to cause a loss of biological activity (i.e.,
does not denature the protein). Exemplary mild detergents include,
but are not limited to, Tween (e.g., Tween-20), Triton (e.g.,
Triton X-100), NP-40 (nonyl phenoxylpolyethoxylethanol), Nonidet
P-40 (octyl phenoxylpolyethoxylethanol), and Sodium Dodecyl Sulfate
(SDS).
Antibody "effector functions" refer to those biological activities
attributable to the Fc region (a native sequence Fc region or amino
acid sequence variant Fc region) of an antibody, and vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g., B
cell receptor); and B cell activation.
"Antibody-dependent cell-mediated cytotoxicity" or "ADCC refers to
a form of cytotoxicity in which secreted Ig bound to Fc receptors
(FcRs) present on certain cytotoxic cells (e.g., Natural Killer
(NK) cells, neutrophils, and macrophages) enable these cytotoxic
effector cells to bind specifically to an antigen-bearing target
cell and subsequently kill the target cell with cytotoxic agents.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No.
5,821,337 can be performed. Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest can be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al., Proc. Natl. Acad.
Sci. USA 95:652-656 (1998).
"Fc receptor" or "FcR" describes a receptor that binds to the Fc
region of an antibody. The preferred FcR is a human FcR. Moreover,
a preferred FcR is one that binds an IgG antibody (a gamma
receptor) and includes receptors of the Fc.gamma.RI, Fc.gamma.RII,
and Fc.gamma.RIII subclasses, including allelic variants and
alternatively spliced forms of these receptors. Fc.gamma.RII
receptors include Fc.gamma.RIIA (an "activating receptor") and
Fc.gamma.RIIB (an "inhibiting receptor"), which have similar amino
acid sequences that differ primarily in the cytoplasmic domains
thereof. Activating receptor Fc.gamma.RIIA contains an
immunoreceptor tyrosine-based activation motif (ITAM) in its
cytoplasmic domain. Inhibiting receptor Fc.gamma.RIIB contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its
cytoplasmic domain (see review M. Daeron, Annu. Rev. Immunol.
15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu.
Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34
(1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).
Other FcRs, including those to be identified in the future, are
encompassed by the term "FcR" herein. The term also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587
(1976) and Kim et al., J. Immunol. 24:249 (1994)).
"Human effector cells" are leukocytes that express one or more FcRs
and perform effector functions. Preferably, the cells express at
least Fc.gamma.RIII and perform ADCC effector function. Examples of
human leukocytes that mediate ADCC include peripheral blood
mononuclear cells (PBMC), natural killer (NK) cells, monocytes,
cytotoxic T cells, and neutrophils; with PBMCs and NK cells being
preferred. The effector cells can be isolated from a native source,
e.g., from blood.
"Complement dependent cytotoxicity" or "CDC refers to the lysis of
a target cell in the presence of complement. Activation of the
classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) that are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be
performed.
The term "therapeutically effective amount" refers to an amount of
an antibody, antibody fragment, or derivative to treat a disease or
disorder in a subject. In the case of tumor (e.g., a cancerous
tumor), the therapeutically effective amount of the antibody or
antibody fragment (e.g., a multispecific antibody or antibody
fragment) may reduce the number of cancer cells; reduce the primary
tumor size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the disorder. To the extent
the antibody or antibody fragment may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic. For
cancer therapy, efficacy in vivo can, for example, be measured by
assessing the duration of survival, time to disease progression
(TTP), the response rates (RR), duration of response, and/or
quality of life.
By "reduce or inhibit" is meant the ability to cause an overall
decrease preferably of 20% or greater, more preferably of 50% or
greater, and most preferably of 75%, 85%, 90%, 95%, or greater.
Reduce or inhibit can refer to the symptoms of the disorder being
treated, the presence or size of metastases, the size of the
primary tumor, or the size or number of the blood vessels in
angiogenic disorders.
The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Included in this
definition are benign and malignant cancers. Examples of cancer
include but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers
include squamous cell cancer, small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of
the lung, cancer of the peritoneum, hepatocellular cancer, gastric
or stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, glioma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer (e.g., renal cell
carcinoma), liver cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, and various types of head and neck cancer. By "early
stage cancer" is meant a cancer that is not invasive or metastatic
or is classified as a Stage 0, I, or II cancer. The term
"precancerous" refers to a condition or a growth that typically
precedes or develops into a cancer. By "non-metastatic" is meant a
cancer that is benign or that remains at the primary site and has
not penetrated into the lymphatic or blood vessel system or to
tissues other than the primary site. Generally, a non-metastatic
cancer is any cancer that is a Stage 0, I, or II cancer, and
occasionally a Stage III cancer.
An "allergic or inflammatory disorder" herein is a disease or
disorder that results from a hyper-activation of the immune system
of an individual. Exemplary allergic or inflammatory disorders
include, but are not limited to, asthma, psoriasis, rheumatoid
arthritis, atopic dermatitis, multiple sclerosis, systemic lupus,
erythematosus, eczema, organ transplantation, age-related macular
degeneration, Crohn's disease, ulcerative colitis, eosinophilic
esophagitis, and autoimmune diseases associated with
inflammation.
An "autoimmune disease" herein is a disease or disorder arising
from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis such as
acute arthritis, chronic rheumatoid arthritis, gouty arthritis,
acute gouty arthritis, chronic inflammatory arthritis, degenerative
arthritis, infectious arthritis, Lyme arthritis, proliferative
arthritis, psoriatic arthritis, vertebral arthritis, and
juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis
chronica progrediente, arthritis deformans, polyarthritis chronica
primaria, reactive arthritis, and ankylosing spondylitis),
inflammatory hyperproliferative skin diseases, psoriasis such as
plaque psoriasis, gutatte psoriasis, pustular psoriasis, and
psoriasis of the nails, dermatitis including contact dermatitis,
chronic contact dermatitis, allergic dermatitis, allergic contact
dermatitis, dermatitis herpetiformis, and atopic dermatitis,
x-linked hyper IgM syndrome, urticaria such as chronic allergic
urticaria and chronic idiopathic urticaria, including chronic
autoimmune urticaria, polymyositis/dermatomyositis, juvenile
dermatomyositis, toxic epidermal necrolysis, scleroderma (including
systemic scleroderma), sclerosis such as systemic sclerosis,
multiple sclerosis (MS) such as spino-optical MS, primary
progressive MS (PPMS), and relapsing remitting MS (RRMS),
progressive systemic sclerosis, atherosclerosis, arteriosclerosis,
sclerosis disseminata, and ataxic sclerosis, inflammatory bowel
disease (IBD) (for example, Crohn's disease, autoimmune-mediated
gastrointestinal diseases, colitis such as ulcerative colitis,
colitis ulcerosa, microscopic colitis, collagenous colitis, colitis
polyposa, necrotizing enterocolitis, and transmural colitis, and
autoimmune inflammatory bowel disease), pyoderma gangrenosum,
erythema nodosum, primary sclerosing cholangitis, episcleritis),
respiratory distress syndrome, including adult or acute respiratory
distress syndrome (ARDS), meningitis, inflammation of all or part
of the uvea, iritis, choroiditis, an autoimmune hematological
disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated
diseases such as anaphylaxis and allergic and atopic rhinitis,
encephalitis such as Rasmussen's encephalitis and limbic and/or
brainstem encephalitis, uveitis, such as anterior uveitis, acute
anterior uveitis, granulomatous uveitis, nongranulomatous uveitis,
phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN or idiopathic membranous nephropathy,
membrano- or membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
allergic reaction, eczema including allergic or atopic eczema,
asthma such as asthma bronchiale, bronchial asthma, and auto-immune
asthma, conditions involving infiltration of T cells and chronic
inflammatory responses, chronic pulmonary inflammatory disease,
autoimmune myocarditis, leukocyte adhesion deficiency, systemic
lupus erythematosus (SLE) or systemic lupus erythematodes such as
cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal
lupus syndrome (NLE), lupus erythematosus disseminatus, lupus
(including nephritis, cerebritis, pediatric, non-renal,
extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes
mellitus, including pediatric insulin-dependent diabetes mellitus
(IDDM), adult onset diabetes mellitus (Type II diabetes),
autoimmune diabetes, idiopathic diabetes insipidus, immune
responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including lymphomatoid granulomatosis, Wegener's
granulomatosis, agranulocytosis, vasculitides, including vasculitis
(including large vessel vasculitis (including polymyalgia
rheumatica and giant cell (Takayasu's) arteritis), medium vessel
vasculitis (including Kawasaki's disease and polyarteritis nodosa),
microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous,
or hypersensitivity vasculitis, systemic necrotizing vasculitis,
and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or
syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
hemolytic anemia or immune hemolytic anemia including autoimmune
hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa),
Addison's disease, pure red cell anemia or aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome such as
those secondary to septicemia, trauma or hemorrhage,
antigen-antibody complex-mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet's or Behcet's disease, Castleman's
syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's
syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid
bullous and skin pemphigoid, pemphigus (including pemphigus
vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid,
and pemphigus erythematosus), autoimmune polyendocrinopathies,
Reiter's disease or syndrome, immune complex nephritis,
antibody-mediated nephritis, neuromyelitis optica,
polyneuropathies, chronic neuropathy such as IgM polyneuropathies
or IgM-mediated neuropathy, thrombocytopenia (as developed by
myocardial infarction patients, for example), including thrombotic
thrombocytopenic purpura (TTP) and autoimmune or immune-mediated
thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP)
including chronic or acute ITP, autoimmune disease of the testis
and ovary including autoimmune orchitis and oophoritis, primary
hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as autoimmune thyroiditis, Hashimoto's
disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, polyglandular syndromes such as autoimmune
polyglandular syndromes (or polyglandular endocrinopathy
syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome
or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,
encephalomyelitis such as allergic encephalomyelitis or
encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE), myasthenia gravis such as
thymoma-associated myasthenia gravis, cerebellar degeneration,
neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS),
and sensory neuropathy, multifocal motor neuropathy, Sheehan's
syndrome, autoimmune hepatitis, chronic hepatitis, lupoid
hepatitis, giant cell hepatitis, chronic active hepatitis or
autoimmune chronic active hepatitis, lymphoid interstitial
pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP,
Guillain-Barre syndrome, Berger's disease (IgA nephropathy),
idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary
cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome,
Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy),
refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic
lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery
disease, autoimmune ear disease such as autoimmune inner ear
disease (AIED), autoimmune hearing loss, opsoclonus myoclonus
syndrome (OMS), polychondritis such as refractory or relapsed
polychondritis, pulmonary alveolar proteinosis, amyloidosis,
scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis,
which includes monoclonal B cell lymphocytosis (e.g., benign
monoclonal gammopathy and monoclonal garnmopathy of undetermined
significance, MGUS), peripheral neuropathy, paraneoplastic
syndrome, channelopathies such as epilepsy, migraine, arrhythmia,
muscular disorders, deafness, blindness, periodic paralysis, and
channelopathies of the CNS, autism, inflammatory myopathy, focal
segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy,
uveoretinitis, chorioretinitis, autoimmune hepatological disorder,
fibromyalgia, multiple endocrine failure, Schmidt's syndrome,
adrenalitis, gastric atrophy, presenile dementia, demyelinating
diseases such as autoimmune demyelinating diseases, diabetic
nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome
(calcinosis, Raynaud's phenomenon, esophageal dysmotility,
sclerodactyl), and telangiectasia), male and female autoimmune
infertility, mixed connective tissue disease, Chagas' disease,
rheumatic fever, recurrent abortion, farmer's lung, erythema
multiforme, post-cardiotomy syndrome, Cushing's syndrome,
bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis,
Sampter's syndrome, Caplan's syndrome, dengue, endocarditis,
endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis,
interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic
fibrosis, endophthalmitis, erythema elevatum et diutinum,
erythroblastosis fetalis, eosinophilic faciitis, Shulman's
syndrome, Felty's syndrome, flariasis, cyclitis such as chronic
cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's
cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus
(HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's
disease, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, giant cell polymyalgia, endocrine ophthamopathy,
chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca,
epidemic keratoconjunctivitis, idiopathic nephritic syndrome,
minimal change nephropathy, benign familial and
ischemia-reperfusion injury, retinal autoimmunity, joint
inflammation, bronchitis, chronic obstructive airway disease,
silicosis, aphthae, aphthous stomatitis, arteriosclerotic
disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica, enteritis allergica, erythema nodosum leprosum,
idiopathic facial paralysis, chronic fatigue syndrome, febris
rheumatica, Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary
idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis
granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma
gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,
infertility due to antispermatozoan antibodies, non-malignant
thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases,
acquired immune deficiency syndrome (AIDS), parasitic diseases such
as Leishmania, toxic-shock syndrome, food poisoning, conditions
involving infiltration of T cells, leukocyte-adhesion deficiency,
immune responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, diseases involving
leukocyte diapedesis, multiple organ injury syndrome,
antigen-antibody complex-mediated diseases, antiglomerular basement
membrane disease, allergic neuritis, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune
atrophic gastritis, sympathetic ophthalmia, rheumatic diseases,
mixed connective tissue disease, nephrotic syndrome, insulitis,
polyendocrine failure, peripheral neuropathy, autoimmune
polyglandular syndrome type I, adult-onset idiopathic
hypoparathyroidism (AOIH), alopecia totalis, dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA),
hemochromatosis, myocarditis, nephrotic syndrome, primary
sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or
chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid
sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,
Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary eosinophilia, bronchopneumonic aspergillosis,
aspergilloma, or granulomas containing eosinophils, anaphylaxis,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune disorders associated with collagen disease, rheumatism,
neurological disease, ischemic re-perfusion disorder, reduction in
blood pressure response, vascular dysfunction, antgiectasis, tissue
injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia,
and disease accompanying vascularization, allergic hypersensitivity
disorders, glomerulonephritides, reperfusion injury, reperfusion
injury of myocardial or other tissues, dermatoses with acute
inflammatory components, acute purulent meningitis or other central
nervous system inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated
syndromes, cytokine-induced toxicity, acute serious inflammation,
chronic intractable inflammation, pyelitis, pneumonocirrhosis,
diabetic retinopathy, diabetic large-artery disorder, endarterial
hyperplasia, peptic ulcer, valvulitis, and endometriosis.
The term "cytotoxic agent" as used herein refers to a substance
that inhibits or prevents the function of a cell and/or causes
destruction of a cell. The term is intended to include radioactive
isotopes (e.g., At.sup.211, I.sup.131, I.sub.125, Y.sup.90,
Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, Ra.sup.223,
P.sup.32, and radioactive isotopes of Lu), chemotherapeutic agents,
e.g., methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor, anticancer, and
chemotherapeutic agents disclosed herein. Other cytotoxic agents
are described herein. A tumoricidal agent causes destruction of
tumor cells.
"chemotherapeutic agent" is a chemical compound useful in the
treatment of cancer. Examples of chemotherapeutic agents include
alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma 1 (see, e.g., Agnew, Chem. Intl. Ed. Engl. 33: 183-186
(1994)); dynemicin, including dynemicin A; an esperamicin; as well
as neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores), aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, carabicin, caminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholine-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
Also included in this definition are anti-hormonal agents that act
to regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer, and are often in the form of
systemic, or whole-body treatment. They may be hormones themselves.
Examples include anti-estrogens and selective estrogen receptor
modulators (SERMs), including, for example, tamoxifen (including
NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene, droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and FARESTON.RTM. toremifene; anti-progesterones; estrogen receptor
down-regulators (ERDs); agents that function to suppress or shut
down the ovaries, for example, leutinizing hormone-releasing
hormone (LHRH) agonists such as LUPRON.RTM. and ELIGARD.RTM.
leuprolide acetate, goserelin acetate, buserelin acetate and
tripterelin; other anti-androgens such as flutamide, nilutamide and
bicalutamide; and aromatase inhibitors that inhibit the enzyme
aromatase, which regulates estrogen production in the adrenal
glands, such as, for example, 4(5)-imidazoles, aminoglutethimide,
MEGASE.RTM. megestrol acetate, AROMASIN.RTM. exemestane,
formestanie, fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM.
letrozole, and ARIMIDEX.RTM. anastrozole. In addition, such
definition of chemotherapeutic agents includes bisphosphonates such
as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
A "growth inhibitory agent" when used herein refers to a compound
or composition which inhibits growth of a cell either in vitro or
in vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (e.g., vincristine and vinblastine), taxanes,
and topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. The agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
"Anti-cancer therapy" as used herein refers to a treatment that
reduces or inhibits cancer in a subject. Examples of anti-cancer
therapy include cytotoxic radiotherapy as well as the
administration of a therapeutically effective amount of a cytotoxic
agent, a chemotherapeutic agent, a growth inhibitory agent, a
cancer vaccine, an angiogenesis inhibitor, a prodrug, a cytokine, a
cytokine antagonist, a corticosteroid, an immunosuppressive agent,
an anti-emetic, an antibody or antibody fragment, or an analgesic
to the subject.
The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). Prodrugs include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs,
beta-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
The term "cytokine" is a generic term for proteins released by one
cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone (HGH), N-methionyl
human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH), and luteinizing hormone (LH);
epidermal growth factor (EGF); hepatic growth factor; fibroblast
growth factor (FGF); prolactin; placental lactogen; tumor necrosis
factor-alpha and -beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, -beta
and -gamma colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF
(G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-1beta,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12; IL-18a tumor necrosis factor such as TNF-alpha or TNF-beta;
and other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cytokine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native sequence cytokines.
By "cytokine antagonist" is meant a molecule that partially or
fully blocks, inhibits, or neutralizes a biological activity of at
least one cytokine. For example, the cytokine antagonists may
inhibit cytokine activity by inhibiting cytokine expression and/or
secretion, or by binding to a cytokine or to a cytokine receptor.
Cytokine antagonists include antibodies, synthetic or
native-sequence peptides, immunoadhesins, and small-molecule
antagonists that bind to a cytokine or cytokine receptor. The
cytokine antagonist is optionally conjugated with or fused to a
cytotoxic agent. Exemplary TNF antagonists are etanercept
(ENBREL.RTM.), infliximab (REMICADE.RTM.), and adalimumab
(HUMIRA.TM.).
The term "immunosuppressive agent" as used herein refers to
substances that act to suppress or mask the immune system of the
subject being treated. This includes substances that suppress
cytokine production, downregulate or suppress self-antigen
expression, or mask the MHC antigens. Examples of immunosuppressive
agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S.
Pat. No. 4,665,077); mycophenolate mofetil such as CELLCEPT.RTM.;
azathioprine (IMURAN.RTM., AZASAN.RTM./6-mercaptopurine;
bromocryptine; danazol; dapsone; glutaraldehyde (which masks the
MHC antigens, as described in U.S. Pat. No. 4,120,649);
anti-idiotypic antibodies for MHC antigens and MHC fragments;
cyclosporin A; steroids such as corticosteroids and
glucocorticosteroids, e.g., prednisone, prednisolone such as
PEDIAPRED.RTM. (prednisolone sodium phosphate) or ORAPRED.RTM.
(prednisolone sodium phosphate oral solution), methylprednisolone,
and dexamethasone; methotrexate (oral or subcutaneous)
(RHEUMATREX.RTM., TREXALL.TM.); hydroxycloroquine/chloroquine;
sulfasalazine; leflunomide; cytokine or cytokine receptor
antagonists including anti-interferon-.gamma., -.beta., or -.alpha.
antibodies, anti-tumor necrosis factor-.alpha. antibodies
(infliximab or adalimumab), anti-TNF.alpha. immunoadhesin
(ENBREL.RTM., etanercept), anti-tumor necrosis factor-.beta.
antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor
antibodies; anti-LFA-1 antibodies, including anti-CD11a and
anti-CD18 antibodies; anti-L3T4 antibodies; heterologous
anti-lymphocyte globulin; polyclonal or pan-T antibodies, or
monoclonal anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide
containing a LFA-3 binding domain (WO 90/08187); streptokinase;
TGF-.beta.; streptodornase; RNA or DNA from the host; FK506;
RS-61443; deoxyspergualin; rapamycin; T-cell receptor (Cohen et
al., U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et
al. Science 251: 430-432 (1991); WO 90/11294; laneway, Nature
341:482 (1989); and WO 91/01133); T cell receptor antibodies (EP
340,109) such as T10B9; cyclophosphamide (CYTOXAN.RTM.); dapsone;
penicillamine (CUPRIMINE.RTM.); plasma exchange; or intravenous
immunoglobulin (IVIG). These may be used alone or in combination
with each other, particularly combinations of steroid and another
immunosuppressive agent or such combinations followed by a
maintenance dose with a non-steroid agent to reduce the need for
steroids.
An "analgesic" refers to a drug that acts to inhibit or suppress
pain in a subject. Exemplary analgesics include non-steroidal
anti-inflammatory drugs (NSAIDs) including ibuprofen (MOTRIN.RTM.),
naproxen (NAPROSYN.RTM.), acetylsalicylic acid, indomethacin,
sulindac, and tolmetin, including salts and derivatives thereof, as
well as various other medications used to reduce the stabbing pains
that may occur, including anticonvulsants (gabapentin, phenyloin,
carbamazepine) or tricyclic antidepressants. Specific examples
include acetaminophen, aspirin, amitriptyline (ELAVIL.RTM.),
carbamazepine (TEGRETOL.RTM.), phenyltoin (DILANTIN.RTM.),
gabapentin (NEURONTIN.RTM.), (E)-N-Vanillyl-8-methyl-6-noneamid
(CAPSAICIN.RTM.), or a nerve blocker.
"Corticosteroid" refers to any one of several synthetic or
naturally occurring substances with the general chemical structure
of steroids that mimic or augment the effects of the naturally
occurring corticosteroids. Examples of synthetic corticosteroids
include prednisone, prednisolone (including methylprednisolone),
dexamethasone triamcinolone, and betamethasone.
A "cancer vaccine," as used herein is a composition that stimulates
an immune response in a subject against a cancer. Cancer vaccines
typically consist of a source of cancer-associated material or
cells (antigen) that may be autologous (from self) or allogenic
(from others) to the subject, along with other components (e.g.,
adjuvants) to further stimulate and boost the immune response
against the antigen. Cancer vaccines can result in stimulating the
immune system of the subject to produce antibodies to one or
several specific antigens, and/or to produce killer T cells to
attack cancer cells that have those antigens.
"Cytotoxic radiotherapy" as used herein refers to radiation therapy
that inhibits or prevents the function of cells and/or causes
destruction of cells. Radiation therapy may include, for example,
external beam irradiation or therapy with a radioactive labeled
agent, such as an antibody. The term is intended to include use of
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212,
Ra.sup.223, P.sup.32, and radioactive isotopes of Lu).
A "subject" is a vertebrate, such as a mammal, e.g., a human.
Mammals include, but are not limited to, farm animals (such as
cows), sport animals, pets (such as cats, dogs and horses),
primates, mice, and rats.
Except where indicated otherwise by context, the terms "first"
polypeptide and "second" polypeptide, and variations thereof, are
merely generic identifiers, and are not to be taken as identifying
a specific or a particular polypeptide or component of antibodies
of the invention.
Commercially available reagents referred to in the Examples were
used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
Examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Unless otherwise noted, the present invention uses standard
procedures of recombinant DNA technology, such as those described
hereinabove and in the following textbooks: Sambrook et al., supra;
Ausubel et al., Current Protocols in Molecular Biology (Green
Publishing Associates and Wiley Interscience, NY, 1989); Innis et
al., PCR Protocols: A Guide to Methods and Applications (Academic
Press, Inc., NY, 1990); Harlow et al., Antibodies: A Laboratory
Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait,
Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney,
Animal Cell Culture, 1987; Coligan et al., Current Protocols in
Immunology, 1991.
Throughout this specification and claims, the word "comprise," or
variations such as "comprises" or "comprising," will be understood
to imply the inclusion of a stated integer or group of integers but
not the exclusion of any other integer or group of integers.
II. CONSTRUCTION OF HETEROMULTIMERIC PROTEINS
Typically, the heteromultimeric proteins described herein will
comprise a significant portion of an antibody Fc region. In other
aspects, however, the heavy chain comprises only a portion of the
C.sub.H1, C.sub.H2, and/or C.sub.H3 domains.
Heteromultimerization Domains
The heteromultimeric proteins comprise a heteromultimerization
domain. To generate a substantially homogeneous population of
heterodimeric molecule, the heterodimerization domain must have a
strong preference for forming heterodimers over homodimers.
Although the heteromultimeric proteins exemplified herein use the
knobs into holes technology to facilitate heteromultimerization
those skilled in the art will appreciate other
heteromultimerization domains useful in the instant invention.
Knobs into Holes
The use of knobs into holes as a method of producing multispecific
antibodies is well known in the art. See U.S. Pat. No. 5,731,168
granted 24 Mar. 1998 assigned to Genentech, PCT Pub. No.
WO2009089004 published 16 Jul. 2009 and assigned to Amgen, and US
Pat. Pub. No. 20090182127 published 16 Jul. 2009 and assigned to
Novo Nordisk A/S. See also Marvin and Zhu, Acta Pharmacologica
Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol.
Sin., 26:1-9. A brief discussion is provided here.
A "protuberance" refers to at least one amino acid side chain which
projects from the interface of a first polypeptide and is therefore
positionable in a compensatory cavity in the adjacent interface
(i.e. the interface of a second polypeptide) so as to stabilize the
heteromultimer, and thereby favor heteromultimer formation over
homomultimer formation, for example. The protuberance may exist in
the original interface or may be introduced synthetically (e.g. by
altering nucleic acid encoding the interface). Normally, nucleic
acid encoding the interface of the first polypeptide is altered to
encode the protuberance. To achieve this, the nucleic acid encoding
at least one "original" amino acid residue in the interface of the
first polypeptide is replaced with nucleic acid encoding at least
one "import" amino acid residue which has a larger side chain
volume than the original amino acid residue. It will be appreciated
that there can be more than one original and corresponding import
residue. The upper limit for the number of original residues which
are replaced is the total number of residues in the interface of
the first polypeptide. The side chain volumes of the various amino
residues are shown in the following table.
TABLE-US-00001 TABLE 1 Properties of Amino Acid Residues Accessible
Surface One-Letter MASS.sup.a VOLUME.sup.b Area.sup.c Amino Acid
Abbreviation (daltons) (Angstrom.sup.3) (Angstrom.sup.2) Alanine
(Ala) A 71.08 88.6 115 Arginine (Arg) R 156.20 173.4 225 Asparagine
(Asn) N 114.11 117.7 160 Aspartic acid D 115.09 111.1 150 (Asp)
Cysteine (Cys) C 103.14 108.5 135 Glutamine (Gln) Q 128.14 143.9
180 Glutamic acid E 129.12 138.4 190 (Glu) Glycine (Gly) G 57.06
60.1 75 Histidine (His) H 137.15 153.2 195 Isoleucine (Ile) I
113.17 166.7 175 Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K
128.18 168.6 200 Methionine (Met) M 131.21 162.9 185 Phenylalinine
F 147.18 189.9 210 (Phe) Proline (Pro) P 97.12 122.7 145 Serine
(Ser) S 87.08 89.0 115 Threonine (Thr) T 101.11 116.1 140
Tryptophan (Trp) W 186.21 227.8 255 Tyrosine (Tyr) Y 163.18 193.6
230 Valine (Val) V 99.14 140.0 155 .sup.aMolecular weight amino
acid minus that of water. Values from Handbook of Chemistry and
Physics, 43rd ed. Cleveland, Chemical Rubber Publishing Co., 1961.
.sup.bValues from A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24:
107-123, 1972. .sup.CValues from C. Chothia, J. Mol. Biol. 105:
1-14, 1975. The accessible surface area is defined in FIG. 6-20 of
this reference.
The preferred import residues for the formation of a protuberance
are generally naturally occurring amino acid residues and are
preferably selected from arginine (R), phenylalanine (F), tyrosine
(Y) and tryptophan (W). Most preferred are tryptophan and tyrosine.
In one embodiment, the original residue for the formation of the
protuberance has a small side chain volume, such as alanine,
asparagine, aspartic acid, glycine, serine, threonine or
valine.
A "cavity" refers to at least one amino acid side chain which is
recessed from the interface of a second polypeptide and therefore
accommodates a corresponding protuberance on the adjacent interface
of a first polypeptide. The cavity may exist in the original
interface or may be introduced synthetically (e.g. by altering
nucleic acid encoding the interface). Normally, nucleic acid
encoding the interface of the second polypeptide is altered to
encode the cavity. To achieve this, the nucleic acid encoding at
least one "original" amino acid residue in the interface of the
second polypeptide is replaced with DNA encoding at least one
"import" amino acid residue which has a smaller side chain volume
than the original amino acid residue. It will be appreciated that
there can be more than one original and corresponding import
residue. The upper limit for the number of original residues which
are replaced is the total number of residues in the interface of
the second polypeptide. The side chain volumes of the various amino
residues are shown in Table 1 above. The preferred import residues
for the formation of a cavity are usually naturally occurring amino
acid residues and are preferably selected from alanine (A), serine
(S), threonine (T) and valine (V). Most preferred are serine,
alanine or threonine. In one embodiment, the original residue for
the formation of the cavity has a large side chain volume, such as
tyrosine, arginine, phenylalanine or tryptophan.
An "original" amino acid residue is one which is replaced by an
"import" residue which can have a smaller or larger side chain
volume than the original residue. The import amino acid residue can
be a naturally occurring or non-naturally occurring amino acid
residue, but preferably is the former. "Naturally occurring" amino
acid residues are those residues encoded by the genetic code and
listed in Table 1 above. By "non-naturally occurring" amino acid
residue is meant a residue which is not encoded by the genetic
code, but which is able to covalently bind adjacent amino acid
residue(s) in the polypeptide chain. Examples of non-naturally
occurring amino acid residues are norleucine, ornithine, norvaline,
homoserine and other amino acid residue analogues such as those
described in Ellman et al., Meth. Enzym. 202:301-336 (1991), for
example. To generate such non-naturally occurring amino acid
residues, the procedures of Noren et al. Science 244: 182 (1989)
and Ellman et al., supra can be used. Briefly, this involves
chemically activating a suppressor tRNA with a non-naturally
occurring amino acid residue followed by in vitro transcription and
translation of the RNA. The method of the instant invention
involves replacing at least one original amino acid residue, but
more than one original residue can be replaced. Normally, no more
than the total residues in the interface of the first or second
polypeptide will comprise original amino acid residues which are
replaced. Typically, original residues for replacement are
"buried". By "buried" is meant that the residue is essentially
inaccessible to solvent. Generally, the import residue is not
cysteine to prevent possible oxidation or mispairing of disulfide
bonds.
The protuberance is "positionable" in the cavity which means that
the spatial location of the protuberance and cavity on the
interface of a first polypeptide and second polypeptide
respectively and the sizes of the protuberance and cavity are such
that the protuberance can be located in the cavity without
significantly perturbing the normal association of the first and
second polypeptides at the interface. Since protuberances such as
Tyr, Phe and Trp do not typically extend perpendicularly from the
axis of the interface and have preferred conformations, the
alignment of a protuberance with a corresponding cavity relies on
modeling the protuberance/cavity pair based upon a
three-dimensional structure such as that obtained by X-ray
crystallography or nuclear magnetic resonance (NMR). This can be
achieved using widely accepted techniques in the art.
By "original or template nucleic acid" is meant the nucleic acid
encoding a polypeptide of interest which can be "altered" (La
genetically engineered or mutated) to encode a protuberance or
cavity. The original or starting nucleic acid may be a naturally
occurring nucleic acid or may comprise a nucleic acid which has
been subjected to prior alteration (e.g. a humanized antibody
fragment). By "altering" the nucleic acid is meant that the
original nucleic acid is mutated by inserting, deleting or
replacing at least one codon encoding an amino acid residue of
interest. Normally, a codon encoding an original residue is
replaced by a codon encoding an import residue. Techniques for
genetically modifying a DNA in this manner have been reviewed in
Mutagenesis: a Practical Approach, M. J. McPherson, Ed., (IRL
Press, Oxford, UK. (1991), and include site-directed mutagenesis,
cassette mutagenesis and polymerase chain reaction (PCR)
mutagenesis, for example. By mutating an original/template nucleic
acid, an original/template polypeptide encoded by the
original/template nucleic acid is thus correspondingly altered.
The protuberance or cavity can be "introduced" into the interface
of a first or second polypeptide by synthetic means, e.g. by
recombinant techniques, in vitro peptide synthesis, those
techniques for introducing non-naturally occurring amino acid
residues previously described, by enzymatic or chemical coupling of
peptides or some combination of these techniques. Accordingly, the
protuberance or cavity which is "introduced" is "non-naturally
occurring" or "non-native", which means that it does not exist in
nature or in the original polypeptide (e.g. a humanized monoclonal
antibody).
Generally, the import amino acid residue for forming the
protuberance has a relatively small number of "rotomers" (e.g.
about 3-6). A "rotomer" is an energetically favorable conformation
of an amino acid side chain. The number of rotomers of the various
amino acid residues are reviewed in Ponders and Richards, J. Mol.
Biol. 193: 775-791 (1987).
III. VECTORS, HOST CELLS AND RECOMBINANT METHODS
For recombinant production of a heteromultimeric protein (e.g., an
antibody) of the invention, the nucleic acid encoding it is
isolated and inserted into a replicable vector for further cloning
(amplification of the DNA) or for expression. DNA encoding the
antibody is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of the antibody). Many vectors are available. The choice of
vector depends in part on the host cell to be used. Generally,
preferred host cells are of either prokaryotic or eukaryotic
(generally mammalian, but also including fungi (e.g., yeast),
insect, plant, and nucleated cells from other multicellular
organisms) origin. It will be appreciated that constant regions of
any isotype can be used for this purpose, including IgG, IgM, IgA,
IgD, and IgE constant regions, and that such constant regions can
be obtained from any human or animal species.
a. Generating Heteromultimeric Proteins Using Prokaryotic Host
Cells
i. Vector Construction
Polynucleotide sequences encoding polypeptide components of the
heteromultimeric proteins (e.g., an antibody) of the invention can
be obtained using standard recombinant techniques. Desired
polynucleotide sequences may be isolated and sequenced from, for
example, antibody producing cells such as hybridoma cells.
Alternatively, polynucleotides can be synthesized using nucleotide
synthesizer or PCR techniques. Once obtained, sequences encoding
the polypeptides are inserted into a recombinant vector capable of
replicating and expressing heterologous polynucleotides in
prokaryotic hosts. Many vectors that are available and known in the
art can be used for the purpose of the present invention. Selection
of an appropriate vector will depend mainly on the size of the
nucleic acids to be inserted into the vector and the particular
host cell to be transformed with the vector. Each vector contains
various components, depending on its function (amplification or
expression of heterologous polynucleotide, or both) and its
compatibility with the particular host cell in which it resides.
The vector components generally include, but are not limited to: an
origin of replication, a selection marker gene, a promoter, a
ribosome binding site (RBS), a signal sequence, the heterologous
nucleic acid insert and a transcription termination sequence.
In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
The expression vector of the invention may comprise two or more
promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. An inducible promoter is a promoter that
initiates increased levels of transcription of the cistron under
its control in response to changes in the culture condition, e.g.,
the presence or absence of a nutrient or a change in
temperature.
A large number of promoters recognized by a variety of potential
host cells are well known. The selected promoter can be operably
linked to cistron DNA encoding, for example, the light or heavy
chain by removing the promoter from the source DNA via restriction
enzyme digestion and inserting the isolated promoter sequence into
the vector of the invention. Both the native promoter sequence and
many heterologous promoters may be used to direct amplification
and/or expression of the target genes. In some embodiments,
heterologous promoters are utilized, as they generally permit
greater transcription and higher yields of the expressed target
gene as compared to the native target polypeptide promoter.
Promoters suitable for use with prokaryotic hosts include the PhoA
promoter, the .beta.-galactamase and lactose promoter systems, a
tryptophan (trp) promoter system and hybrid promoters such as the
tac or the trc promoter. However, other promoters that are
functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker to operably
ligate them to cistrons encoding the genes of the heteromultimeric
protein, e.g., the target light and heavy chains (Siebenlist et
al., (1980) Cell 20: 269), using linkers or adaptors to supply any
required restriction sites.
In one aspect of the invention, each cistron within the recombinant
vector comprises a secretion signal sequence component that directs
translocation of the expressed polypeptides across a membrane. In
general, the signal sequence may be a component of the vector, or
it may be a part of the target polypeptide DNA that is inserted
into the vector. The signal sequence selected for the purpose of
this invention should be one that is recognized and processed
(i.e., cleaved by a signal peptidase) by the host cell. For
prokaryotic host cells that do not recognize and process the signal
sequences native to the heterologous polypeptides, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group consisting of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders,
LamB, PhoE, PeIB, OmpA and MBP. In one embodiment of the invention,
the signal sequences used in both cistrons of the expression system
are STII signal sequences or variants thereof.
In another aspect, the production of the immunoglobulins according
to the invention can occur in the cytoplasm of the host cell, and
therefore does not require the presence of secretion signal
sequences within each cistron. In that regard, immunoglobulin light
and heavy chains are expressed, folded and assembled to form
functional immunoglobulins within the cytoplasm. Certain host
strains (e.g., the E. coli trxff strains) provide cytoplasm
conditions that are favorable for disulfide bond formation, thereby
permitting proper folding and assembly of expressed protein
subunits. See Proba and Pluckthun Gene, 159:203 (1995).
Prokaryotic host cells suitable for expressing heteromultimeric
proteins (e.g., antibodies) of the invention include Archaebacteria
and Eubacteria, such as Gram-negative or Gram-positive organisms.
Examples of useful bacteria include Escherichia (e.g., E. coli),
Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species
(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhuA (.DELTA.ton) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP41 kan.sup.R (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli.sub..lamda. 1776 (ATCC
31,537) and E. coli RV308 (ATCC 31,608) are also suitable. In one
embodiment, E. coli .DELTA.lpp finds particular use. These examples
are illustrative rather than limiting. Methods for constructing
derivatives of any of the above-mentioned bacteria having defined
genotypes are known in the art and described in, for example, Bass
et al., Proteins, 8:309-314 (1990). It is generally necessary to
select the appropriate bacteria taking into consideration
replicability of the replicon in the cells of a bacterium. For
example, E. coli, Serratia, or Salmonella species can be suitably
used as the host when well known plasmids such as pBR322, pBR325,
pACYC177, or pKN410 are used to supply the replicon. Typically the
host cell should secrete minimal amounts of proteolytic enzymes,
and additional protease inhibitors may desirably be incorporated in
the cell culture.
ii. Polypeptide Production
Host cells are transformed with the above-described expression
vectors and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences.
Transformation means introducing DNA into the prokaryotic host so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
Prokaryotic cells used to produce the polypeptides of the invention
are grown in media known in the art and suitable for culture of the
selected host cells. Examples of suitable media include Luria broth
(LB) plus necessary nutrient supplements. In some embodiments, the
media also contains a selection agent, chosen based on the
construction of the expression vector, to selectively permit growth
of prokaryotic cells containing the expression vector. For example,
ampicillin is added to media for growth of cells expressing
ampicillin resistant gene.
Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
The prokaryotic host cells are cultured at suitable temperatures.
For E. coli growth, for example, the preferred temperature ranges
from about 20.degree. C. to about 39.degree. C., more preferably
from about 25.degree. C. to about 37.degree. C., even more
preferably at about 30.degree. C. The pH of the medium may be any
pH ranging from about 5 to about 9, depending mainly on the host
organism. For E. coli, the pH is preferably from about 6.8 to about
7.4, and more preferably about 7.0.
If an inducible promoter is used in the expression vector of the
invention, protein expression is induced under conditions suitable
for the activation of the promoter. In one aspect of the invention,
PhoA promoters are used for controlling transcription of the
polypeptides. Accordingly, the transformed host cells are cultured
in a phosphate-limiting medium for induction. Preferably, the
phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons
et al., J. Immunol. Methods (2002), 263:133-147). A variety of
other inducers may be used, according to the vector construct
employed, as is known in the art.
In one embodiment, the first and second hinge-containing host cells
are cultured separately and the expressed polypeptides of the
present invention are secreted into and recovered from the
periplasm of the host cells separately. In a second embodiment, the
first and second hinge-containing host cells are cultured
separately and prior to the isolation of the hinge-containing
polypeptides, the two host cell cultures are mixed together and the
cells pelleted. In a third embodiment, the first and second
hinge-containing host cells are cultured separately, centrifuged
and resuspended separately and then mixed together prior to
isolation of the hinge-containing polypeptides. In fourth
embodiment, the first and second hinge-containing host cells are
cultured together in the same culture vessel. Protein recovery
typically involves disrupting the microorganism cell membrane,
generally by such means as osmotic shock, sonication or lysis. Once
cells are disrupted, cell debris or whole cells may be removed by
centrifugation or filtration. The proteins may be further purified,
for example, by affinity resin chromatography. Alternatively,
proteins can be transported into the culture media and isolated
therein. Cells may be removed from the culture and the culture
supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay. The isolated polypeptides will be used to produce the
heteromultimeric proteins at In one aspect of the invention,
heteromultimeric protein (e.g., antibody) production is conducted
in large quantity by a fermentation process. Various large-scale
fed-batch fermentation procedures are available for production of
recombinant proteins. Large-scale fermentations have at least 1000
liters of capacity, preferably about 1,000 to 100,000 liters of
capacity. These fermentors use agitator impellers to distribute
oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
In a fermentation process, induction of protein expression is
typically initiated after the cells have been grown under suitable
conditions to a desired density, e.g., an OD.sub.550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
To improve the production yield and quality of the polypeptides of
the invention, various fermentation conditions can be modified. For
example, to improve the proper assembly and folding of the secreted
heteromultimeric proteins (e.g., antibodies), additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), Proc. Natl. Acad. Sci. USA
95:2773-2777; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et
al., U.S. Pat. No. 5,508,192; Nara et al., Microbial Drug
Resistance, 2:63-72 (1996).
In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention. In a second embodiment, the E. coli strain is
deficient for a lipoprotein of the outer membrane (.DELTA.lpp).
iii. Heteromultimeric Protein Purification
In one embodiment, the heteromultimeric protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed.
The following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
In one aspect, Protein A immobilized on a solid phase is used for
immunoaffinity purification of, for example, full length antibody
products of the invention. Protein A is a 41 kD cell wall protein
from Staphylococcus aureus which binds with a high affinity to the
Fc region of antibodies. Lindmark et al. (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
As the first step of purification, the preparation derived from the
cell culture as described above is applied onto the Protein A
immobilized solid phase to allow specific binding of the antibody
of interest to Protein A. The solid phase is then washed to remove
contaminants non-specifically bound to the solid phase. The
heteromultimeric protein (e.g., antibody) is recovered from the
solid phase by elution.
b. Generating Heteromultimeric Proteins Using Eukaryotic Host
Cells:
The vector components generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
i. Signal Sequence Component
A vector for use in a eukaryotic host cell may also contain a
signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected preferably is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available. The DNA for
such precursor region is ligated in reading frame to DNA encoding
the desired heteromultimeric protein(s) (e.g., antibodies).
ii. Origin of Replication
Generally, an origin of replication component is not needed for
mammalian expression vectors. For example, the SV40 origin may
typically be used, but only because it contains the early
promoter.
iii. Selection Gene Component
Expression and cloning vectors may contain a selection gene, also
termed a selectable marker. Typical selection genes encode proteins
that (a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, where relevant, or (c) supply critical
nutrients not available from complex media.
One example of a selection scheme utilizes a drug to arrest growth
of a host cell. Those cells that are successfully transformed with
a heterologous gene produce a protein conferring drug resistance
and thus survive the selection regimen. Examples of such dominant
selection use the drugs neomycin, mycophenolic acid and
hygromycin.
Another example of suitable selectable markers for mammalian cells
are those that enable the identification of cells competent to take
up the antibody nucleic acid, such as DHFR, thymidine kinase,
metallothionein-I and -II, preferably primate metallothionein
genes, adenosine deaminase, ornithine decarboxylase, etc.
For example, cells transformed with the DHFR selection gene are
first identified by culturing all of the transformants in a culture
medium that contains methotrexate (Mtx), a competitive antagonist
of DHFR. An appropriate host cell when wild-type DHFR is employed
is the Chinese hamster ovary (CHO) cell line deficient in DHFR
activity (e.g., ATCC CRL-9096).
Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418.
See, for example, U.S. Pat. No. 4,965,199.
iv. Promoter Component
Expression and cloning vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the
desired hinge-containing polypeptide(s) (e.g., antibody) nucleic
acid. Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
Desired hinge-containing polypeptide(s) (e.g., antibody)
transcription from vectors in mammalian host cells is controlled,
for example, by promoters obtained from the genomes of viruses such
as, for example, polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus
40 (SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, or from heat-shock
promoters, provided such promoters are compatible with the host
cell systems.
The early and late promoters of the SV40 virus are conveniently
obtained as an SV40 restriction fragment that also contains the
SV40 viral origin of replication. The immediate early promoter of
the human cytomegalovirus is conveniently obtained as a HindIII E
restriction fragment. A system for expressing DNA in mammalian
hosts using the bovine papilloma virus as a vector is disclosed in
U.S. Pat. No. 4,419,446. A modification of this system is described
in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature
297:598-601 (1982) on expression of human .beta.-interferon cDNA in
mouse cells under the control of a thymidine kinase promoter from
herpes simplex virus. Alternatively, the Rous Sarcoma Virus long
terminal repeat can be used as the promoter.
v. Enhancer Element Component
Transcription of DNA encoding the desired hinge-containing
polypeptide(s) (e.g., antibody) by higher eukaryotes can be
increased by inserting an enhancer sequence into the vector. Many
enhancer sequences are now known from mammalian genes (e.g.,
globin, elastase, albumin, .alpha.-fetoprotein, and insulin genes).
Also, one may use an enhancer from a eukaryotic cell virus.
Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18
(1982) for a description of elements for enhancing activation of
eukaryotic promoters. The enhancer may be spliced into the vector
at a position 5' or 3' to the antibody polypeptide-encoding
sequence, provided that enhancement is achieved, but is generally
located at a site 5' from the promoter.
vi. Transcription Termination Component
Expression vectors used in eukaryotic host cells will typically
also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
vii. Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
Host cells are transformed with the above-described expression or
cloning vectors for desired hinge-containing polypeptide(s) (e.g.,
antibody) production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
viii. Culturing the Host Cells
The host cells used to produce a desired hinge-containing
polypeptide(s) (e.g., antibody) of this invention may be cultured
in a variety of media. Commercially available media such as Ham's
F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the
media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et
al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704;
4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO
87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for
the host cells. Any of these media may be supplemented as necessary
with hormones and/or other growth factors (such as insulin,
transferrin, or epidermal growth factor), salts (such as sodium
chloride, calcium, magnesium, and phosphate), buffers (such as
HEPES), nucleotides (such as adenosine and thymidine), antibiotics
(such as GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
ix. Purification of Heteromultimeric Proteins
When using recombinant techniques, the hinge-containing
polypeptides can be produced intracellularly, or directly secreted
into the medium. If the hinge-containing polypeptide is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Where the hinge-containing
polypeptide is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
The heteromultimer composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25 M salt). The
production of the heteromultimeric proteins can alternatively or
additionally (to any of the foregoing particular methods) comprise
dialyzing a solution comprising a mixture of the polypeptides.
x. Antibody Production Using Baculovirus
Recombinant baculovirus may be generated by co-transfecting a
plasmid encoding an antibody or antibody fragment and
BaculoGold.TM. virus DNA (Pharmingen) into an insect cell such as a
Spodoptera frugiperda cell (e.g., Sf9 cells; ATCC CRL 1711) or a
Drosophila melanogaster S2 cell using, for example, lipofectin
(commercially available from GIBCO-BRL). In a particular example,
an antibody sequence is fused upstream of an epitope tag contained
within a baculovirus expression vector. Such epitope tags include
poly-His tags. A variety of plasmids may be employed, including
plasmids derived from commercially available plasmids such as
pVL1393 (Novagen) or pAcGP67B (Pharmingen). Briefly, the sequence
encoding an antibody or a fragment thereof may be amplified by PCR
with primers complementary to the 5' and 3' regions. The 5' primer
may incorporate flanking (selected) restriction enzyme sites. The
product may then be digested with the selected restriction enzymes
and subcloned into the expression vector.
After transfection with the expression vector, the host cells
(e.g., Sf9 cells) are incubated for 4-5 days at 28.degree. C. and
the released virus is harvested and used for further
amplifications. Viral infection and protein expression may be
performed as described, for example, by O'Reilley et al.
(Baculovirus expression vectors: A Laboratory Manual. Oxford:
Oxford University Press (1994)).
Expressed poly-His tagged antibody can then be purified, for
example, by Ni2+-chelate affinity chromatography as follows.
Extracts can be prepared from recombinant virus-infected Sf9 cells
as described by Rupert et al. (Nature 362:175-179 (1993)). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL HEPES
pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40;
0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate; 300 mM NaCl;
10% glycerol pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni2+-NTA agarose column (commercially available from Qiagen) is
prepared with a bed volume of 5 mL, washed with 25 mL of water, and
equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl; 10% glycerol
pH 6.0), which elutes nonspecifically bound protein. After reaching
A280 baseline again, the column is developed with a 0 to 500 mM
Imidazole gradient in the secondary wash buffer. One mL fractions
are collected and analyzed by SDS-PAGE and silver staining or
Western blot with Ni2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His10-tagged antibody are
pooled and dialyzed against loading buffer.
Alternatively, purification of the antibody can be performed using
known chromatography techniques, including for instance, Protein A
or protein G column chromatography. In one embodiment, the antibody
of interest may be recovered from the solid phase of the column by
elution into a solution containing a chaotropic agent or mild
detergent. Exemplary chaotropic agents and mild detergents include,
but are not limited to, Guanidine-HCl, urea, lithium perclorate,
Arginine, Histidine, SDS (sodium dodecyl sulfate), Tween, Triton,
and NP-40, all of which are commercially available.
IV. HETEROMULTIMERIC PROTEIN FORMATION/ASSEMBLY
The formation of the complete heteromultimeric protein involves the
reassembly of the first and second hinge-containing polypeptides by
disulfide bond formation which in the present invention is referred
to as refolding. Refolding includes the association of the first
hinge-containing polypeptide with the second hinge-containing
polypeptide and the formation of the interchain disulfide bonds.
Refolding, also termed renaturing, in the present invention is done
in vitro without the addition of reductant.
The host cells may be cultured using the above described methods
either as separate cultures or as a single culture. In one method,
the first host cells and second host cells are grown in the same
culture vessel (sometimes referred to herein as co-cultured or a
mixed culture). In another method, the first and second host cells
are grown in separate culture vessels. In one method, the separate
cultures are processed separately then mixed/combined prior to
disruption of the cellular membrane. In another method, the
separate cultures are mixed then processed prior to disruption of
the cellular membrane. In one method, the separate cultures are
mixed without further processing prior to disruption of the
cellular membrane. In one method, the single culture comprising the
first and second host cells is processed prior to disruption of the
cellular membrane. In another method, the co-cultured cells are not
processed prior to disruption of the cellular membrane. Processing
of the cells comprises centrifugation and resuspension in an
appropriate buffer (e.g., extraction buffer).
Extraction buffers are known in the art and the skilled artisan
will be able to determine which buffer to use without undue
experimentation.
The host cell membranes are disrupted using methods known in the
art. Such methods include cell membrane permeablization and cell
membrane disintegration. Permeablizing the cell membrane refers to
rendering the membrane "leaky", e.g., by introducing holes, without
destroying the overall integrity of the membrane such that the cell
remains viable. In other words, permeabilization provides
macromolecular movement across the cellular membrane and preserves
cellular structure sufficiently to allow continued cell viability.
In contrast, cell membrane disintegration results in the cellular
contents being released into the extracellular milieu and cell
death.
Methods for disrupting cell membranes include but are not limited
to enzymatic lysis. sonication, osmotic shock, passage through a
microfluidizer, addition of EDTA, use various detergents, solvents
(such as toluene, dimethyl sulfoxide, etc), surfactants (such as
Triton-X 100, Tween 20, etc), hypotonic buffers, use of freeze/thaw
techniques, electroporation, and passage through a stainless steel
ball homogenizer.
Once the hinge-containing polypeptides are released from the cell
(either by permeabilization or disintegration) the
heteromultimerization domains will drive the association of the
heteromultimeric proteins. Inter-chain disulfide formation of the
associated hinge-containing polypeptides proceeds without the
addition of reducing agents. The resultant disulfide linked
heteromultimeric protein is then purified. Optionally, it may be
formulated for research, diagnostic, therapeutic or other
purposes.
V. TARGET MOLECULES
Examples of molecules that may be targeted by a heteromultimeric
protein of this invention include, but are not limited to, soluble
serum proteins and their receptors and other membrane bound
proteins (e.g., adhesins).
In another embodiment the heteromultimeric protein of the invention
is capable of binding one, two or more cytokines, cytokine-related
proteins, and cytokine receptors selected from the group consisting
of BMPI, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, CSFI (M-CSF), CSF2
(GM-CSF), CSF3 (G-CSF), EPO, FGFI (aFGF), FGF2 (bFGF), FGF3
(int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10,
FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21,
FGF23, IGF1, IGF2, IFNAI, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNBI,
IFNG, IFNWI, FELI, FELI (EPSELON), FELI (ZETA), IL1A, IL1B, IL2,
IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13,
IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24,
IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA,
TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40
ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand),
TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSFI0 (TRAIL),
TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B,
TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF,
VEGFB, VEGFC, ILIR1, IL1R2, IL1RL1, LL1RL2, IL2RA, IL2RB, IL2RG,
IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, ILI0RA, ILI0RB,
IL1IRA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1,
IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN,
IL6ST, IL18BP, IL18RAP, IL22RA2, AIFI, HGF, LEP (leptin), PTN, and
THPO.
In another embodiment, a target molecule is a chemokine, chemokine
receptor, or a chemokine-related protein selected from the group
consisting of CCLI (I-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-Ia), CCL4
(MIP-Ib), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLH
(eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17
(TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3a), CCL21
(SLC/exodus-2), CCL22 (MDC/STC-I), CCL23 (MPIF-I), CCL24
(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27
(CTACK/ILC), CCL28, CXCLI (GROI), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5
(ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP 10), CXCL11
(I-TAC), CXCL12 (SDFI), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP
(CXCL7), CX3CL1 (SCYDI), SCYEI, XCLI (lymphotactin), XCL2 (SCM-Ib),
BLRI (MDR15), CCBP2 (D6/JAB61), CCRI (CKRI/HM145), CCR2
(mcp-IRB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6
(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBII), CCR8
(CMKBR8/TERI/CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHKI), CCRL2 (L-CCR),
XCRI (GPR5/CCXCRI), CMKLRI, CMKORI (RDCI), CX3CR1 (V28), CXCR4,
GPR2 (CCRI0), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6
(TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R
(GPR16), TOPIO, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6,
CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCCIO (CIO), EPO, FY (DARC),
GDF5, HDFIA, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREMI,
TREM2, and VHL.
In another embodiment the heteromultimeric proteins of the
invention are capable of binding one or more targets selected from
the group consisting of ABCFI; ACVRI; ACVRIB; ACVR2; ACVR2B;
ACVRLI; AD0RA2A; Aggrecan; AGR2; AICDA; AIFI; AIGI; AKAPI; AKAP2;
AMH; AMHR2; ANGPTI; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOCI;
AR; AZGPI (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys);
BAGI; BAII; BCL2; BCL6; BDNF; BLNK; BLRI (MDR15); BMPI; BMP2; BMP3B
(GDFIO); BMP4; BMP6; BMP8; BMPRIA; BMPRIB; BMPR2; BPAGI (plectin);
BRCAI; C19orfIO (IL27w); C3; C4A; C5; C5R1; CANTI; CASP1; CASP4;
CAVI; CCBP2 (D6/JAB61); CCLI (1-309); CCLII (eotaxin); CCL13
(MCP-4); CCL15 (MIP-Id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC);
CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MTP-2);
SLC; exodus-2; CCL22 (MDC/STC-I); CCL23 (MPIF-1); CCL24
(MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27
(CTACK/ILC); CCL28; CCL3 (MTP-1a); CCL4 (MDP-Ib); CCL5 (RANTES);
CCL7 (MCP-3); CCL8 (mcp-2); CCNAI; CCNA2; CCNDI; CCNEI; CCNE2; CCRI
(CKRI/HM145); CCR2 (mcp-IRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5
(CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7
(CKR7/EBII); CCR8 (CMKBR8/TERI/CKR-LI); CCR9 (GPR-9-6); CCRLI
(VSHKI); CCRL2 (L-CCR); CD164; CD19; CDIC; CD20; CD200; CD22; CD24;
CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44;
CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81;
CD83; CD86; CDHI (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19;
CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7;
CDK9; CDKNIA (p21Wapl/Cipl); CDKNIB (p27Kipl); CDKNIC; CDKN2A
(P16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CERI; CHGA; CHGB;
Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6;
CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin);
CMKLRI; CMKORI (RDCI); CNRI; COL18A1; COLIAI; COL4A3; COL6A1; CR2;
CRP; CSFI (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNBI
(b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDI); CX3CR1 (V28);
CXCLI (GROI); CXCL10 (IP-10); CXCLII (1-TAC/IP-9); CXCL12 (SDFI);
CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5
(ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2);
CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYCI; CYSLTRI; DAB2IP;
DES; DKFZp451J0118; DNCLI; DPP4; E2F1; ECGFI; EDGI; EFNAI; EFNA3;
EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2
(Her-2); EREG; ERK8; ESRI; ESR2; F3 (TF); FADD; FasL; FASN; FCERIA;
FCER2; FCGR3A; FGF; FGFI (aFGF); FGF10; FGF11; FGF12; FGF12B;
FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20;
FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2);
FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FELI (EPSILON); FILI
(ZETA); FLJ12584; FLJ25530; FLRTI (fibronectin); FLTI; FOS; FOSLI
(FRA-I); FY (DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-6ST;
GATA3; GDF5; GFI1; GGT1; GM-CSF; GNASI; GNRHI; GPR2 (CCRIO); GPR31;
GPR44; GPR81 (FKSG80); GRCCIO (CIO); GRP; GSN (Gelsolin); GSTPI;
HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIFIA; HDPI; histamine
and histamine receptors; HLA-A; HLA-DRA; HM74; HMOXI; HUMCYT2A;
ICEBERG; ICOSL; ID2; IFN-a; IFNAI; IFNA2; IFNA4; IFNA5; IFNA6;
IFNA7; IFNB1; IFNgamma; DFNWI; IGBPI; IGFI; IGFIR; IGF2; IGFBP2;
IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12;
IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15;
IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1;
IL18RAP; IL19; IL1A, IL1B; ILIF10; IL1F5; IL1F6; IL1F7; IL1F8;
IL1F9; IL1HYI; IL1RI; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1;
IL1RL2, ILIRN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2;
IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB;
IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST
(glycoprotein 130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R;
DLK; INHA; INHBA; INSL3; INSL4; IRAKI; ERAK2; ITGAI; ITGA2; ITGA3;
ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAGI;
JAK1; JAK3; JUN; K6HF; KAII; KDR; KITLG; KLF5 (GC Box BP); KLF6;
KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9;
KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6 (hair-specific type H
keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA
(TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp;
MAP2K7 (c-Jun); MDK; MIBI; midkine; MEF; MIP-2; MKI67; (Ki-67);
MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSSI; MUCI
(mucin); MYC; MYD88; NCK2; neurocan; NFKBI; NFKB2; NGFB (NGF);
NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NMEI
(NM23A); N0X5; NPPB; NROBI; NR0B2; NRIDI; NR1D2; NR1H2; NR1H3;
NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2;
NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1;
NRPI; NRP2; NT5E; NTN4; ODZI; OPRDI; P2RX7; PAP; PARTI; PATE; PAWR;
PCA3; PCNA; PDGFA; PDGFB; PECAMI; PF4 (CXCL4); PGF; PGR;
phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDCI; PPBP (CXCL7);
PPID; PRI; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN;
PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGSI; RGS13; RGS3; RNFIIO
(ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1
(mammaglobin2); SCGB2A2 (mammaglobin 1); SCYEI (endothelial
Monocyte-activating cytokine); SDF2; SERPINAI; SERPINA3; SERP1NB5
(maspin); SERPINEI (PAI-I); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1;
SLC43A1; SLIT2; SPPI; SPRRIB (Sprl); ST6GAL1; STABI; STAT6; STEAP;
STEAP2; TB4R2; TBX21; TCPIO; TDGFI; TEK; TGFA; TGFBI; TGFBIII;
TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; THIL; THBSI
(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissue
factor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF;
TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSFIA; TNFRSFIB;
TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9;
TNFSFIO (TRAIL); TNFSFI 1 (TRANCE); TNFSF12 (APO3L); TNFSF13
(April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18;
TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7
(CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1 BB ligand);
TOLLIP; Toll-like receptors; TOP2A (topoisomerase Ea); TP53; TPMI;
TPM2; TRADD; TRAFI; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREMI;
TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5;
VLA-4; XCLI (lymphotactin); XCL2 (SCM-Ib); XCRI (GPR5/CCXCRI); YYI;
and ZFPM2.
Preferred molecular target molecules for antibodies encompassed by
the present invention include CD proteins such as CD3, CD4, CD8,
CD16, CD19, CD20, CD34; CD64, CD200 members of the ErbB receptor
family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell
adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1,
VCAM, alpha4/beta7 integrin, and alphav/beta3 integrin including
either alpha or beta subunits thereof (e.g., anti-CD11a, anti-CD18
or anti-CD11b antibodies); growth factors such as VEGF-A, VEGF-C;
tissue factor (TF); alpha interferon alphaIFN); TNFalpha, an
interleukin, such as IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-9, IL-13,
IL17A/F, IL-18, IL-13Ralpha1, IL13Ralpha2, IL-4R, IL-5R, IL-9R,
IgE; blood group antigens; flk2/flt3 receptor; obesity (OB)
receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein, protein
C etc.
In one embodiment, the heteromultimeric proteins of this invention
bind low-density lipoprotein receptor-related protein (LRP)-1 or
LRP-8 or transferrin receptor, and at least one target selected
from the group consisting of 1) beta-secretase (BACE1 or BACE2), 2)
alpha-secretase, 3) gamma-secretase, 4) tau-secretase, 5) amyloid
precursor protein (APP), 6) death receptor 6 (DR6), 7) amyloid beta
peptide, 8) alpha-synuclein, 9) Parkin, 10) Huntingtin, 11) p75
NTR, and 12) caspase-6.
In one embodiment, the heteromultimeric proteins of this invention
binds to at least two target molecules selected from the group
consisting of: IL-1alpha and IL-1beta, IL-12 and IL-18; IL-13 and
IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and
IL-1beta; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and
MEF; IL-13 and TGF-.beta.; IL-13 and LHR agonist; IL-12 and TWEAK,
IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and
ADAM8, IL-13 and PED2, IL17A and IL17F, CD3 and CD19, CD138 and
CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38 and CD138;
CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CD20
and BR3, TNFalpha and TGF-beta, TNFalpha and IL-1beta; TNFalpha and
IL-2, TNF alpha and IL-3, TNFalpha and IL-4, TNFalpha and IL-5,
TNFalpha and IL6, TNFalpha and IL8, TNFalpha and IL-9, TNFalpha and
IL-10, TNFalpha and IL-11, TNFalpha and IL-12, TNFalpha and IL-13,
TNFalpha and IL-14, TNFalpha and IL-15, TNFalpha and IL-16,
TNFalpha and IL-17, TNFalpha and IL-18, TNFalpha and IL-19,
TNFalpha and IL-20, TNFalpha and IL-23, TNFalpha and IFNalpha,
TNFalpha and CD4, TNFalpha and VEGF, TNFalpha and MIF, TNFalpha and
ICAM-1, TNFalpha and PGE4, TNFalpha and PEG2, TNFalpha and RANK
ligand, TNFalpha and Te38; TNFalpha and BAFF; TNFalpha and CD22;
TNFalpha and CTLA-4; TNFalpha and GP130; TNF.alpha. and IL-12p40;
VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2,
VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8,
VEGF and MET, VEGFR and MET receptor, VEGFR and EGFR, HER2 and
CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR(HER1) and
HER2, EGFR and HER3, EGFR and HER4, IL-13 and CD40L, IL4 and CD40L,
TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and
CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and
BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B; MAG and RGM A; NgR and RGM
A; NogoA and RGM A; OMGp and RGM A; PDL-I and CTLA-4; and RGM A and
RGM B.
Soluble antigens or fragments thereof, optionally conjugated to
other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g., the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g., cancer cell lines) or may
be cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
VI. ACTIVITY ASSAYS
The heteromultimeric proteins of the present invention can be
characterized for their physical/chemical properties and biological
functions by various assays known in the art.
The purified heteromultimeric proteins can be further characterized
by a series of assays including, but not limited to, N-terminal
sequencing, amino acid analysis, non-denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange chromatography and papain digestion.
In certain embodiments of the invention, the immunoglobulins
produced herein are analyzed for their biological activity. In some
embodiments, the immunoglobulins of the present invention are
tested for their antigen binding activity. The antigen binding
assays that are known in the art and can be used herein include,
without limitation, any direct or competitive binding assays using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays. An illustrative antigen binding assay is provided
below in the Examples section.
In one embodiment, the present invention contemplates an altered
antibody that possesses some but not all effector functions, which
make it a desired candidate for many applications in which the half
life of the antibody in vivo is important yet certain effector
functions (such as complement and ADCC) are unnecessary or
deleterious. In certain embodiments, the Fc activities of the
produced heteromultimeric protein are measured to ensure that only
the desired properties are maintained. In vitro and/or in vivo
cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
heteromultimeric protein lacks Fc.gamma.R binding (hence likely
lacking ADCC activity), but retains FcRn binding ability. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). An example of an in vitro assay to assess ADCC
activity of a molecule of interest is described in U.S. Pat. No.
5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
natural killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998). C1q binding assays may also be carried out
to confirm that the antibody is unable to bind C1q and hence lacks
CDC activity. To assess complement activation, a CDC assay, e.g. as
described in Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996), may be performed. FcRn binding and in vivo clearance/half
life determinations can also be performed using methods known in
the art.
VII. CONJUGATED PROTEINS
The invention also provides conjugated proteins such as conjugated
antibodies or immunoconjugates (for example, "antibody-drug
conjugates" or "ADC"), comprising any of the heteromultimeric
proteins described herein (e.g., an antibody made according to the
methods described herein) where one of the constant regions of the
light chain or the heavy chain is conjugated to a chemical molecule
such as a dye or cytotoxic agent such as a chemotherapeutic agent,
a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate). In particular, as described herein, the use of
heteromultimerization domains enables the construction of
antibodies containing two different heavy chains (HC1 and HC2) as
well as two different light chains (LC1 and LC2). An
immunoconjugate constructed using the methods described herein may
contain the cytotoxic agent conjugated to a constant region of only
one of the heavy chains (HC1 or HC2) or only one of the light
chains (LC1 or LC2). Also, because the immunoconjugate can have the
cytotoxic agent attached to only one heavy or light chain, the
amount of the cytotoxic agent being administered to a subject is
reduced relative to administration of an antibody having the
cytotoxic agent attached to both heavy or light chains. Reducing
the amount of cytotoxic agent being administered to a subject
limits adverse side effects associated with the cytotoxic
agent.
The use of antibody-drug conjugates for the local delivery of
cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos,
Anticancer Research 19:605-614 (1999); Niculescu-Duvaz and
Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No.
4,975,278) allows targeted delivery of the drug moiety to tumors,
and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., Lancet (Mar.
15, 1986):603-605 (1986); Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (eds.), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., Cancer Immunol. Immunother. 21:183-187 (1986)).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al., Jour. of the Nat. Cancer
Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic &
Med. Chem. Letters 10:1025-1028 (2000); Mandler et al.,
Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213;
Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and
calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et
al., Cancer Res. 53:3336-3342 (1993)). The toxins may effect their
cytotoxic and cytostatic effects by mechanisms including tubulin
binding, DNA binding, or topoisomerase inhibition. Some cytotoxic
drugs tend to be inactive or less active when conjugated to large
antibodies or protein receptor ligands.
Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein (e.g., above). Enzymatically
active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes. See,
e.g., WO 93/21232 published Oct. 28, 1993. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See, e.g., WO94/11026.
Conjugates of an antibody and one or more small molecule toxins,
such as a calicheamicin, maytansinoids, dolastatins, aurostatins, a
trichothecene, and CC1065, and the derivatives of these toxins that
have toxin activity, are also contemplated herein.
i. Maytansine and Maytansinoids
In some embodiments, the immunoconjugate comprises an antibody
(full length or fragments) of the invention conjugated to one or
more maytansinoid molecules.
Maytansinoids are mitototic inhibitors which act by inhibiting
tubulin polymerization. Maytansine was first isolated from the east
African shrub Maytenus serrata (U.S. Pat. No. 3,896,111).
Subsequently, it was discovered that certain microbes also produce
maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S.
Pat. No. 4,151,042). Synthetic maytansinol and derivatives and
analogues thereof are disclosed, for example, in U.S. Pat. Nos.
4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;
4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;
4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;
4,450,254; 4,362,663; and 4,371,533.
Maytansinoid drug moieties are attractive drug moieties in antibody
drug conjugates because they are: (i) relatively accessible to
prepare by fermentation or chemical modification, derivatization of
fermentation products, (ii) amenable to derivatization with
functional groups suitable for conjugation through the
non-disulfide linkers to antibodies, (iii) stable in plasma, and
(iv) effective against a variety of tumor cell lines.
Immunoconjugates containing maytansinoids, methods of making same,
and their therapeutic use are disclosed, for example, in U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the
disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Antibody-maytansinoid conjugates are prepared by chemically linking
an antibody to a maytansinoid molecule without significantly
diminishing the biological activity of either the antibody or the
maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (the
disclosure of which is hereby expressly incorporated by reference).
An average of 3-4 maytansinoid molecules conjugated per antibody
molecule has shown efficacy in enhancing cytotoxicity of target
cells without negatively affecting the function or solubility of
the antibody, although even one molecule of toxin/antibody would be
expected to enhance cytotoxicity over the use of naked antibody.
Maytansinoids are well known in the art and can be synthesized by
known techniques or isolated from natural sources. Suitable
maytansinoids are disclosed, for example, in U.S. Pat. No.
5,208,020 and in the other patents and nonpatent publications
referred to hereinabove. Preferred maytansinoids are maytansinol
and maytansinol analogues modified in the aromatic ring or at other
positions of the maytansinol molecule, such as various maytansinol
esters.
There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1,
Chari et al., Cancer Research 52:127-131 (1992), and U.S. Patent
Application Publication No. 2005/0169933, the disclosures of which
are hereby expressly incorporated by reference.
Antibody-maytansinoid conjugates comprising the linker component
SMCC may be prepared as disclosed in U.S. Patent Application
Publication No. 2005/0169933. The linking groups include disulfide
groups, thioether groups, acid labile groups, photolabile groups,
peptidase labile groups, or esterase labile groups, as disclosed in
the above-identified patents, disulfide and thioether groups being
preferred. Additional linking groups are described and exemplified
herein.
Conjugates of the antibody and maytansinoid may be made using a
variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 (1978)) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
The linker may be attached to the maytansinoid molecule at various
positions, depending on the type of the link. For example, an ester
linkage may be formed by reaction with a hydroxyl group using
conventional coupling techniques. The reaction may occur at the C-3
position having a hydroxyl group, the C-14 position modified with
hydroxymethyl, the C-15 position modified with a hydroxyl group,
and the C-20 position having a hydroxyl group. In a preferred
embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
ii. Auristatins and Dolastatins
In some embodiments, the immunoconjugate comprises an antibody of
the invention conjugated to dolastatins or dolostatin peptidic
analogs and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483
and 5,780,588). Dolastatins and auristatins have been shown to
interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al., Antimicrob. Agents and
Chemother. 45(12):3580-3584 (2001)) and have anticancer (U.S. Pat.
No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob.
Agents Chemother. 42:2961-2965 (1998)). The dolastatin or
auristatin drug moiety may be attached to the antibody through the
N-(amino) terminus or the C-(carboxyl) terminus of the peptidic
drug moiety (WO 02/088172).
Exemplary auristatin embodiments include the N-terminus linked
monomethylauristatin drug moieties DE and DF, disclosed in
"Monomethylvaline Compounds Capable of Conjugation to Ligands,"
U.S. Application Publication No. 2005/0238649, the disclosure of
which is expressly incorporated by reference in its entirety.
Typically, peptide-based drug moieties can be prepared by forming a
peptide bond between two or more amino acids and/or peptide
fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides," volume 1, pp. 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. Nos. 5,635,483 and 5,780,588; Pettit et
al., J. Nat. Prod. 44:482-485 (1981); Pettit et al., Anti-Cancer
Drug Design 13:47-66 (1998); Poncet, Curr. Pharm. Des. 5:139-162
(1999); and Pettit, Fortschr. Chem. Org. Naturst. 70:1-79 (1997).
See also Doronina, Nat. Biotechnol. 21(7):778-784 (2003); and
"Monomethylvaline Compounds Capable of Conjugation to Ligands,"
U.S. Application Publication No. 2005/0238649, hereby incorporated
by reference in its entirety (disclosing, e.g., linkers and methods
of preparing monomethylvaline compounds such as MMAE and MMAF
conjugated to linkers).
iii. Calicheamicin
In other embodiments, the immunoconjugate comprises an antibody of
the invention conjugated to one or more calicheamicin molecules.
The calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid
Company). Structural analogues of calicheamicin which may be used
include, but are not limited to, .gamma..sub.1.sup.I,
.alpha..sub.2.sup.I, .alpha..sub.3.sup.I,
N-acetyl-.gamma..sub.1.sup.I, PSAG and .theta..sup.I.sub.1 (Hinman
et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer
Research 58:2925-2928 (1998) and the aforementioned U.S. patents to
American Cyanamid). Another anti-tumor drug that the antibody can
be conjugated is QFA, which is an antifolate. Both calicheamicin
and QFA have intracellular sites of action and do not readily cross
the plasma membrane. Therefore, cellular uptake of these agents
through antibody mediated internalization greatly enhances their
cytotoxic effects.
iv. Other Cytotoxic Agents
Other antitumor agents that can be conjugated to the antibodies of
the invention or made according to the methods described herein
include BCNU, streptozoicin, vincristine and 5-fluorouracil, the
family of agents known collectively LL-E33288 complex described in
U.S. Pat. Nos. 5,053,394 and 5,770,710, as well as esperamicins
(U.S. Pat. No. 5,877,296).
Enzymatically active toxins and fragments thereof which can be used
include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes (see, for example, WO 93/21232, published Oct. 28,
1993).
The present invention further contemplates an immunoconjugate
formed between an antibody and a compound with nucleolytic activity
(e.g., a ribonuclease or a DNA endonuclease such as a
deoxyribonuclease; DNase).
For selective destruction of a tumor, the antibody may comprise a
highly radioactive atom. A variety of radioactive isotopes are
available for the production of radioconjugated antibodies.
Examples include At.sup.211, I.sup.131, I.sup.125, Y.sup.90,
Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
The radio- or other labels may be incorporated in the conjugate in
known ways. For example, the peptide may be biosynthesized or may
be synthesized by chemical amino acid synthesis using suitable
amino acid precursors involving, for example, fluorine-19 in place
of hydrogen. Labels such as tc.sup.99m or I.sup.123, Re.sup.186,
Re.sup.188 and In.sup.111 can be attached via a cysteine residue in
the peptide. Yttrium-90 can be attached via a lysine residue. The
IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun.
80:49-57 (1978)) can be used to incorporate iodine-123. "Monoclonal
Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989)
describes other methods in detail.
Conjugates of the antibody and cytotoxic agent may be made using a
variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See, e.g., WO94/11026. The linker
may be a "cleavable linker" facilitating release of the cytotoxic
drug in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Research
52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
The compounds of the invention expressly contemplate, but are not
limited to, ADC prepared with cross-linker reagents: BMPS, EMCS,
GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,
sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
v. Preparation of Conjugated Antibodies
In the conjugated antibodies of the invention, an antibody is
conjugated to one or more moieties (for example, drug moieties),
e.g., about 1 to about 20 moieties per antibody, optionally through
a linker. The conjugated antibodies may be prepared by several
routes, employing organic chemistry reactions, conditions, and
reagents known to those skilled in the art, including: (1) reaction
of a nucleophilic group of an antibody with a bivalent linker
reagent via a covalent bond, followed by reaction with a moiety of
interest; and (2) reaction of a nucleophilic group of a moiety with
a bivalent linker reagent via a covalent bond, followed by reaction
with the nucleophilic group of an antibody. Additional methods for
preparing conjugated antibodies are described herein.
The linker reagent may be composed of one or more linker
components. Exemplary linker components include 6-maleimidocaproyl
("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC`), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").
Additional linker components are known in the art and some are
described herein. See also "Monomethylvaline Compounds Capable of
Conjugation to Ligands," U.S. Application Publication No.
2005/0238649, the contents of which are hereby incorporated by
reference in its entirety.
In some embodiments, the linker may comprise amino acid residues.
Exemplary amino acid linker components include a dipeptide, a
tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides
include: valine-citrulline (vc or val-cit), alanine-phenylalanine
(af or ala-phe). Exemplary tripeptides include:
glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine
(gly-gly-gly). Amino acid residues which comprise an amino acid
linker component include those occurring naturally, as well as
minor amino acids and non-naturally occurring amino acid analogs,
such as citrulline. Amino acid linker components can be designed
and optimized in their selectivity for enzymatic cleavage by a
particular enzymes, for example, a tumor-associated protease,
cathepsin B, C and D, or a plasmin protease.
Nucleophilic groups on antibodies include, but are not limited to:
(i) N-terminal amine groups, (ii) side chain amine groups, e.g.,
lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv)
sugar hydroxyl or amino groups where the antibody is glycosylated.
Amine, thiol, and hydroxyl groups are nucleophilic and capable of
reacting to form covalent bonds with electrophilic groups on linker
moieties and linker reagents including: (i) active esters such as
NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl
and benzyl halides such as haloacetamides; (iii) aldehydes,
ketones, carboxyl, and maleimide groups. Certain antibodies have
reducible interchain disulfides, i.e., cysteine bridges. Antibodies
may be made reactive for conjugation with linker reagents by
treatment with a reducing agent such as DTT (dithiothreitol). Each
cysteine bridge will thus form, theoretically, two reactive thiol
nucleophiles. Additional nucleophilic groups can be introduced into
antibodies through the reaction of lysines with 2-iminothiolane
(Traut's reagent) resulting in conversion of an amine into a thiol.
Reactive thiol groups may be introduced into the antibody (or
fragment thereof) by introducing one, two, three, four, or more
cysteine residues (e.g., preparing mutant antibodies comprising one
or more non-native cysteine amino acid residues).
Conjugated antibodies of the invention may also be produced by
modification of the antibody to introduce electrophilic moieties,
which can react with nucleophilic substituents on the linker
reagent or drug or other moiety. The sugars of glycosylated
antibodies may be oxidized, e.g., with periodate oxidizing
reagents, to form aldehyde or ketone groups which may react with
the amine group of linker reagents or drug or other moieties. The
resulting imine Schiff base groups may form a stable linkage, or
may be reduced, e.g., by borohydride reagents to form stable amine
linkages. In one embodiment, reaction of the carbohydrate portion
of a glycosylated antibody with either glactose oxidase or sodium
meta-periodate may yield carbonyl (aldehyde and ketone) groups in
the protein that can react with appropriate groups on the drug or
other moiety (Hermanson, Bioconjugate Techniques). In another
embodiment, proteins containing N-terminal serine or threonine
residues can react with sodium meta-periodate, resulting in
production of an aldehyde in place of the first amino acid
(Geoghegan and Stroh, Bioconjugate Chem. 3:138-146 (1992); U.S.
Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
Likewise, nucleophilic groups on a moiety (such as a drug moiety)
include, but are not limited to: amine, thiol, hydroxyl, hydrazide,
oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; and (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate. In
yet another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the individual, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
VIII. UTILITY
The present methods provided for herein find industrial
applicability in the production of heteromultimeric proteins. The
inventive methods reduce the amount of work involved in two
separate fermentation and isolations as are technical difficulties
inherent in two separate fermentations. Furthermore, elimination of
the annealment and redox steps of the prior methods procedures can
increase yields and decrease processing complexity and costs.
The heteromultimeric proteins described herein find use in, for
example, in vitro, ex vivo and in vivo therapeutic methods. The
invention provides various methods based on using one or more of
these molecules. In certain pathological conditions, it is
necessary and/or desirable to utilize heteromultimeric proteins,
e.g., multispecific antibodies. The invention provides these
heteromultimeric proteins, which can be used for a variety of
purposes, for example as therapeutics, prophylactics and
diagnostics. For example, the invention provides methods of
treating a disease, said methods comprising administering to a
subject in need of treatment a heteromultimeric protein of the
invention, whereby the disease is treated. Any of the
heteromultimeric proteins of the invention described herein can be
used in therapeutic (or prophylactic or diagnostic) methods
described herein.
For example, when the heteromultimeric protein is multivalent, a
valuable benefit is the enhanced avidity they pose for their
antigen. In addition to having intrinsic high affinity on a binding
unit (ie, a Fab) to antigen basis, normal IgG antibodies also
exploit the avidity effect to increase their association with
antigens as a result of their bivalent binding towards the
targets.
A heteromultimeric protein directed against two separate epitopes
on the same antigen molecule may not only provide the benefit of
enhanced binding avidity (because of bivalent binding), but may
also acquire novel properties that are not associated with either
of the parent antibodies. Thus, the heteromultimeric proteins of
the invention find use in, for example, the blocking of
receptor-ligand interactions.
The heteromultimeric proteins described herein also find use in the
application of simultaneously blocking the signaling pathways of
two targets with one molecule.
IX. THERAPEUTIC USES
The heteromultimeric proteins such as antibodies and antibody
fragments described herein (e.g., an antibody and/or fragment
thereof made according to the methods described herein) may be used
for therapeutic applications. For example, such heteromultimeric
proteins can be used for the treatment of tumors, including
pre-cancerous, non-metastatic, metastatic, and cancerous tumors
(e.g., early stage cancer), for the treatment of allergic or
inflammatory disorders, or for the treatment of autoimmune disease,
or for the treatment of a subject at risk for developing cancer
(for example, breast cancer, colorectal cancer, lung cancer, renal
cell carcinoma, glioma, or ovarian cancer), an allergic or
inflammatory disorder, or an autoimmune disease.
The term cancer embraces a collection of proliferative disorders,
including but not limited to pre-cancerous growths, benign tumors,
and malignant tumors. Benign tumors remain localized at the site of
origin and do not have the capacity to infiltrate, invade, or
metastasize to distant sites. Malignant tumors will invade and
damage other tissues around them. They can also gain the ability to
break off from where they started and spread to other parts of the
body (metastasize), usually through the bloodstream or through the
lymphatic system where the lymph nodes are located. Primary tumors
are classified by the type of tissue from which they arise;
metastatic tumors are classified by the tissue type from which the
cancer cells are derived. Over time, the cells of a malignant tumor
become more abnormal and appear less like normal cells. This change
in the appearance of cancer cells is called the tumor grade and
cancer cells are described as being well-differentiated,
moderately-differentiated, poorly-differentiated, or
undifferentiated. Well-differentiated cells are quite normal
appearing and resemble the normal cells from which they originated.
Undifferentiated cells are cells that have become so abnormal that
it is no longer possible to determine the origin of the cells.
The tumor can be a solid tumor or a non-solid or soft tissue tumor.
Examples of soft tissue tumors include leukemia (e.g., chronic
myelogenous leukemia, acute myelogenous leukemia, adult acute
lymphoblastic leukemia, acute myelogenous leukemia, mature B-cell
acute lymphoblastic leukemia, chronic lymphocytic leukemia,
polymphocytic leukemia, or hairy cell leukemia), or lymphoma (e.g.,
non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's
disease). A solid tumor includes any cancer of body tissues other
than blood, bone marrow, or the lymphatic system. Solid tumors can
be further separated into those of epithelial cell origin and those
of non-epithelial cell origin. Examples of epithelial cell solid
tumors include tumors of the gastrointestinal tract, colon, breast,
prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral
cavity, stomach, duodenum, small intestine, large intestine, anus,
gall bladder, labium, nasopharynx, skin, uterus, male genital
organ, urinary organs, bladder, and skin. Solid tumors of
non-epithelial origin include sarcomas, brain tumors, and bone
tumors.
Epithelial cancers generally evolve from a benign tumor to a
preinvasive stage (e.g., carcinoma in situ), to a malignant cancer,
which has penetrated the basement membrane and invaded the
subepithelial stroma.
Multispecific protein complexes can also be used in these
therapeutic applications, and antibodies that bind HER2 can in
particular be used to treat breast cancer, colorectal cancer, lung
cancer, renal cell carcinoma, glioma, or ovarian cancer.
Other subjects that are candidates for receiving compositions of
this invention have, or are at risk for developing, abnormal
proliferation of fibrovascular tissue, acne rosacea, acquired
immune deficiency syndrome, artery occlusion, atopic keratitis,
bacterial ulcers, Bechets disease, blood borne tumors, carotid
obstructive disease, choroidal neovascularization, chronic
inflammation, chronic retinal detachment, chronic uveitis, chronic
vitritis, contact lens overwear, corneal graft rejection, corneal
neovascularization, corneal graft neovascularization, Crohn's
disease, Eales disease, epidemic keratoconjunctivitis, fungal
ulcers, Herpes simplex infections, Herpes zoster infections,
hyperviscosity syndromes, Kaposi's sarcoma, leukemia, lipid
degeneration, Lyme's disease, marginal keratolysis, Mooren ulcer,
Mycobacteria infections other than leprosy, myopia, ocular
neovascular disease, optic pits, Osler-Weber syndrome
(Osler-Weber-Rendu), osteoarthritis, Paget's disease, pars
planitis, pemphigoid, phylectenulosis, polyarteritis, post-laser
complications, protozoan infections, pseudoxanthoma elasticum,
pterygium keratitis sicca, radial keratotomy, retinal
neovascularization, retinopathy of prematurity, retrolental
fibroplasias, sarcoid, scleritis, sickle cell anemia, Sogren's
syndrome, solid tumors, Stargart's disease, Steven's Johnson
disease, superior limbic keratitis, syphilis, systemic lupus,
Terrien's marginal degeneration, toxoplasmosis, tumors of Ewing
sarcoma, tumors of neuroblastoma, tumors of osteosarcoma, tumors of
retinoblastoma, tumors of rhabdomyosarcoma, ulcerative colitis,
vein occlusion, Vitamin A deficiency, Wegener's sarcoidosis,
undesired angiogenesis associated with diabetes, parasitic
diseases, abnormal wound healing, hypertrophy following surgery,
injury or trauma (e.g., acute lung injury/ARDS), inhibition of hair
growth, inhibition of ovulation and corpus luteum formation,
inhibition of implantation, and inhibition of embryo development in
the uterus.
Examples of allergic or inflammatory disorders or autoimmune
diseases or disorders that may be treated using an antibody made
according to the methods described herein include, but are not
limited to arthritis (rheumatoid arthritis such as acute arthritis,
chronic rheumatoid arthritis, gouty arthritis, acute gouty
arthritis, chronic inflammatory arthritis, degenerative arthritis,
infectious arthritis, Lyme arthritis, proliferative arthritis,
psoriatic arthritis, vertebral arthritis, and juvenile-onset
rheumatoid arthritis, osteoarthritis, arthritis chronica
progrediente, arthritis deformans, polyarthritis chronica primaria,
reactive arthritis, and ankylosing spondylitis), inflammatory
hyperproliferative skin diseases, psoriasis such as plaque
psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of
the nails, dermatitis including contact dermatitis, chronic contact
dermatitis, allergic dermatitis, allergic contact dermatitis,
dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM
syndrome, urticaria such as chronic allergic urticaria and chronic
idiopathic urticaria, including chronic autoimmune urticaria,
polymyositis/dermatomyositis, juvenile dermatomyositis, toxic
epidermal necrolysis, scleroderma (including systemic scleroderma),
sclerosis such as systemic sclerosis, multiple sclerosis (MS) such
as spino-optical MS, primary progressive MS (PPMS), and relapsing
remitting MS (RRMS), progressive systemic sclerosis,
atherosclerosis, arteriosclerosis, sclerosis disseminata, and
ataxic sclerosis, inflammatory bowel disease (IBD) (for example,
Crohn's disease, autoimmune-mediated gastrointestinal diseases,
colitis such as ulcerative colitis, colitis ulcerosa, microscopic
colitis, collagenous colitis, colitis polyposa, necrotizing
enterocolitis, and transmural colitis, and autoimmune inflammatory
bowel disease), pyoderma gangrenosum, erythema nodosum, primary
sclerosing cholangitis, episcleritis), respiratory distress
syndrome, including adult or acute respiratory distress syndrome
(ARDS), meningitis, inflammation of all or part of the uvea,
iritis, choroiditis, an autoimmune hematological disorder,
rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases
such as anaphylaxis and allergic and atopic rhinitis, encephalitis
such as Rasmussen's encephalitis and limbic and/or brainstem
encephalitis, uveitis, such as anterior uveitis, acute anterior
uveitis, granulomatous uveitis, nongranulomatous uveitis,
phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN or idiopathic membranous nephropathy,
membrano- or membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
allergic reaction, eczema including allergic or atopic eczema,
asthma such as asthma bronchiale, bronchial asthma, and auto-immune
asthma, conditions involving infiltration of T-cells and chronic
inflammatory responses, chronic pulmonary inflammatory disease,
autoimmune myocarditis, leukocyte adhesion deficiency, systemic
lupus erythematosus (SLE) or systemic lupus erythematodes such as
cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal
lupus syndrome (NLE), lupus erythematosus disseminatus, lupus
(including nephritis, cerebritis, pediatric, non-renal,
extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes
mellitus, including pediatric insulin-dependent diabetes mellitus
(IDDM), adult onset diabetes mellitus (Type II diabetes),
autoimmune diabetes, idiopathic diabetes insipidus, immune
responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including lymphomatoid granulomatosis, Wegener's
granulomatosis, agranulocytosis, vasculitides, including vasculitis
(including large vessel vasculitis (including polymyalgia
rheumatica and giant cell (Takayasu's) arteritis), medium vessel
vasculitis (including Kawasaki's disease and polyarteritis nodosa),
microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous,
or hypersensitivity vasculitis, systemic necrotizing vasculitis,
and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or
syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
hemolytic anemia or immune hemolytic anemia including autoimmune
hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa),
Addison's disease, pure red cell anemia or aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome such as
those secondary to septicemia, trauma or hemorrhage,
antigen-antibody complex-mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet's or Behcet's disease, Castleman's
syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's
syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid
bullous and skin pemphigoid, pemphigus (including pemphigus
vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid,
and pemphigus erythematosus), autoimmune polyendocrinopathies,
Reiter's disease or syndrome, immune complex nephritis,
antibody-mediated nephritis, neuromyelitis optica,
polyneuropathies, chronic neuropathy such as IgM polyneuropathies
or IgM-mediated neuropathy, thrombocytopenia (as developed by
myocardial infarction patients, for example), including thrombotic
thrombocytopenic purpura (TTP) and autoimmune or immune-mediated
thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP)
including chronic or acute ITP, autoimmune disease of the testis
and ovary including autoimune orchitis and oophoritis, primary
hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as autoimmune thyroiditis, Hashimoto's
disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, polyglandular syndromes such as autoimmune
polyglandular syndromes (or polyglandular endocrinopathy
syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome
or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,
encephalomyelitis such as allergic encephalomyelitis or
encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE), myasthenia gravis such as
thymoma-associated myasthenia gravis, cerebellar degeneration,
neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS),
and sensory neuropathy, multifocal motor neuropathy, Sheehan's
syndrome, autoimmune hepatitis, chronic hepatitis, lupoid
hepatitis, giant cell hepatitis, chronic active hepatitis or
autoimmune chronic active hepatitis, lymphoid interstitial
pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP,
Guillain-Barre syndrome, Berger's disease (IgA nephropathy),
idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary
cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome,
Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy),
refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic
lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery
disease, autoimmune ear disease such as autoimmune inner ear
disease (AIED), autoimmune hearing loss, opsoclonus myoclonus
syndrome (OMS), polychondritis such as refractory or relapsed
polychondritis, pulmonary alveolar proteinosis, amyloidosis,
scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis,
which includes monoclonal B cell lymphocytosis (e.g., benign
monoclonal gammopathy and monoclonal garnmopathy of undetermined
significance, MGUS), peripheral neuropathy, paraneoplastic
syndrome, channelopathies such as epilepsy, migraine, arrhythmia,
muscular disorders, deafness, blindness, periodic paralysis, and
channelopathies of the CNS, autism, inflammatory myopathy, focal
segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy,
uveoretinitis, chorioretinitis, autoimmune hepatological disorder,
fibromyalgia, multiple endocrine failure, Schmidt's syndrome,
adrenalitis, gastric atrophy, presenile dementia, demyelinating
diseases such as autoimmune demyelinating diseases, diabetic
nephropathy, Dressler's syndrome, alopecia areata, CREST syndrome
(calcinosis, Raynaud's phenomenon, esophageal dysmotility,
sclerodactyly, and telangiectasia), male and female autoimmune
infertility, mixed connective tissue disease, Chagas' disease,
rheumatic fever, recurrent abortion, farmer's lung, erythema
multiforme, post-cardiotomy syndrome, Cushing's syndrome,
bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis,
Sampter's syndrome, Caplan's syndrome, dengue, endocarditis,
endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis,
interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic
fibrosis, endophthalmitis, erythema elevatum et diutinum,
erythroblastosis fetalis, eosinophilic faciitis, Shulman's
syndrome, Felty's syndrome, flariasis, cyclitis such as chronic
cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's
cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus
(HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's
disease, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, giant cell polymyalgia, endocrine ophthamopathy,
chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca,
epidemic keratoconjunctivitis, idiopathic nephritic syndrome,
minimal change nephropathy, benign familial and
ischemia-reperfusion injury, retinal autoimmunity, joint
inflammation, bronchitis, chronic obstructive airway disease,
silicosis, aphthae, aphthous stomatitis, arteriosclerotic
disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica, enteritis allergica, erythema nodosum leprosum,
idiopathic facial paralysis, chronic fatigue syndrome, febris
rheumatica, Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary
idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis
granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma
gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,
infertility due to antispermatozoan antobodies, non-malignant
thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases,
acquired immune deficiency syndrome (AIDS), parasitic diseases such
as Leishmania, toxic-shock syndrome, food poisoning, conditions
involving infiltration of T-cells, leukocyte-adhesion deficiency,
immune responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, diseases involving
leukocyte diapedesis, multiple organ injury syndrome,
antigen-antibody complex-mediated diseases, antiglomerular basement
membrane disease, allergic neuritis, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune
atrophic gastritis, sympathetic ophthalmia, rheumatic diseases,
mixed connective tissue disease, nephrotic syndrome, insulitis,
polyendocrine failure, peripheral neuropathy, autoimmune
polyglandular syndrome type I, adult-onset idiopathic
hypoparathyroidism (AOIH), alopecia totalis, dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA),
hemochromatosis, myocarditis, nephrotic syndrome, primary
sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or
chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid
sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,
Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary eosinophilia, bronchopneumonic aspergillosis,
aspergilloma, or granulomas containing eosinophils, anaphylaxis,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune disorders associated with collagen disease, rheumatism,
neurological disease, ischemic re-perfusion disorder, reduction in
blood pressure response, vascular dysfunction, antgiectasis, tissue
injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia,
and disease accompanying vascularization, allergic hypersensitivity
disorders, glomerulonephritides, reperfusion injury, reperfusion
injury of myocardial or other tissues, dermatoses with acute
inflammatory components, acute purulent meningitis or other central
nervous system inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated
syndromes, cytokine-induced toxicity, acute serious inflammation,
chronic intractable inflammation, pyelitis, pneumonocirrhosis,
diabetic retinopathy, diabetic large-artery disorder, endarterial
hyperplasia, peptic ulcer, valvulitis, and endometriosis.
In addition to therapeutic uses, the antibodies of the invention
can be used for other purposes, including diagnostic methods, such
as diagnostic methods for the diseases and conditions described
herein.
X. DOSAGES, FORMULATIONS, AND DURATION
The proteins of this invention will be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual subject, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The "therapeutically effective
amount" of the proteins to be administered will be governed by such
considerations, and is the minimum amount necessary to prevent,
ameliorate, or treat a particular disorder (for example, a cancer,
allergic or inflammatory disorder, or autoimmune disorder). The
proteins need not be, but are optionally, formulated with one or
more agents currently used to prevent or treat the disorder. The
effective amount of such other agents depends on the amount of
proteins present in the formulation, the type of disorder or
treatment, and other factors discussed above. These are generally
used in the same dosages and with administration routes as used
hereinbefore or about from 1 to 99% of the heretofore employed
dosages. Generally, alleviation or treatment of a cancer involves
the lessening of one or more symptoms or medical problems
associated with the cancer. The therapeutically effective amount of
the drug can accomplish one or a combination of the following:
reduce (by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100% or more) the number of cancer cells; reduce or inhibit the
tumor size or tumor burden; inhibit (i.e., to decrease to some
extent and/or stop) cancer cell infiltration into peripheral
organs; reduce hormonal secretion in the case of adenomas; reduce
vessel density; inhibit tumor metastasis; reduce or inhibit tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. In some embodiments, the proteins are
used to prevent the occurrence or reoccurrence of cancer or an
autoimmune disorder in the subject.
In one embodiment, the present invention can be used for increasing
the duration of survival of a human subject susceptible to or
diagnosed with a cancer or autoimmune disorder. Duration of
survival is defined as the time from first administration of the
drug to death. Duration of survival can also be measured by
stratified hazard ratio (HR) of the treatment group versus control
group, which represents the risk of death for a subject during the
treatment.
In yet another embodiment, the treatment of the present invention
significantly increases response rate in a group of human subjects
susceptible to or diagnosed with a cancer who are treated with
various anti-cancer therapies. Response rate is defined as the
percentage of treated subjects who responded to the treatment. In
one aspect, the combination treatment of the invention using
proteins of this invention and surgery, radiation therapy, or one
or more chemotherapeutic agents significantly increases response
rate in the treated subject group compared to the group treated
with surgery, radiation therapy, or chemotherapy alone, the
increase having a Chi-square p-value of less than 0.005. Additional
measurements of therapeutic efficacy in the treatment of cancers
are described in U.S. Patent Application Publication No.
20050186208.
Therapeutic formulations are prepared using standard methods known
in the art by mixing the active ingredient having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences
(20.sup.th edition), ed. A. Gennaro, 2000, Lippincott, Williams
& Wilkins, Philadelphia, Pa.). Acceptable carriers, include
saline, or buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagines, arginine or lysine; monosaccharides, disaccharides,
and other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM., PLURONICS.TM., or PEG.
Optionally, but preferably, the formulation contains a
pharmaceutically acceptable salt, preferably sodium chloride, and
preferably at about physiological concentrations. Optionally, the
formulations of the invention can contain a pharmaceutically
acceptable preservative. In some embodiments the preservative
concentration ranges from 0.1 to 2.0%, typically v/v. Suitable
preservatives include those known in the pharmaceutical arts.
Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben
are preferred preservatives. Optionally, the formulations of the
invention can include a pharmaceutically acceptable surfactant at a
concentration of 0.005 to 0.02%.
The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
The active ingredients may also be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semipermeable matrices of
solid hydrophobic polymers containing the heteromultimeric protein,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated heteromultimeric
protein(s) remain in the body for a long time, they may denature or
aggregate as a result of exposure to moisture at 37.degree. C.,
resulting in a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
The proteins described herein (e.g., a heteromultimeric protein
such as a multispecific antibody made according to the methods
described herein) are administered to a human subject, in accord
with known methods, such as intravenous administration as a bolus
or by continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
Local administration may be particularly desired if extensive side
effects or toxicity is associated with antagonism to the target
molecule recognized by the proteins. An ex vivo strategy can also
be used for therapeutic applications. Ex vivo strategies involve
transfecting or transducing cells obtained from the subject with a
polynucleotide encoding a protein of this invention. The
transfected or transduced cells are then returned to the subject.
The cells can be any of a wide range of types including, without
limitation, hemopoietic cells (e.g., bone marrow cells,
macrophages, monocytes, dendritic cells, T cells, or B cells),
fibroblasts, epithelial cells, endothelial cells, keratinocytes, or
muscle cells.
In one example, the protein complex is (e.g., a heteromultimeric
protein such as a multispecific antibody made according to the
methods described herein) is administered locally, e.g., by direct
injections, when the disorder or location of the tumor permits, and
the injections can be repeated periodically. The protein complex
can also be delivered systemically to the subject or directly to
the tumor cells, e.g., to a tumor or a tumor bed following surgical
excision of the tumor, in order to prevent or reduce local
recurrence or metastasis.
XI. ARTICLES OF MANUFACTURE
Another embodiment of the invention is an article of manufacture
containing one or more protein complexes described herein, and
materials useful for the treatment or diagnosis of a disorder (for
example, an autoimmune disease or cancer). The article of
manufacture comprises a container and a label or package insert on
or associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition that is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is a heteromultimeric protein
(e.g., an antibody or antibody fragment) of the invention. The
label or package insert indicates that the composition is used for
treating the particular condition. The label or package insert will
further comprise instructions for administering the
heteromultimeric protein composition to the subject. Articles of
manufacture and kits comprising combinatorial therapies described
herein are also contemplated.
Package insert refers to instructions customarily included in
commercial packages of therapeutic products that contain
information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such
therapeutic products. In certain embodiments, the package insert
indicates that the composition is used for treating breast cancer,
colorectal cancer, lung cancer, renal cell carcinoma, glioma, or
ovarian cancer.
Additionally, the article of manufacture may further comprise a
second container comprising a pharmaceutically-acceptable buffer,
such as bacteriostatic water for injection (BWFI),
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials considered from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, and syringes.
Kits are also provided that are useful for various purposes, e.g.,
for purification or immunoprecipitation of an antigen (e.g., HER2
or EGFR) from cells. For isolation and purification of an antigen
(e.g., HER2 or EGFR) the kit can contain a heteromultimeric protein
(e.g., an EGFR/HER2 antibody) coupled to beads (e.g., sepharose
beads). Kits can be provided which contain the heteromultimeric
protein(s) for detection and quantitation of the antigen in vitro,
e.g., in an ELISA or a Western blot. As with the article of
manufacture, the kit comprises a container and a label or package
insert on or associated with the container. The container holds a
composition comprising at least one heteromultimeric protein (e.g.,
multispecific antibody or antibody fragment) of the invention.
Additional containers may be included that contain, e.g., diluents
and buffers or control antibodies. The label or package insert may
provide a description of the composition as well as instructions
for the intended in vitro or diagnostic use.
The foregoing written description is considered to be sufficient to
enable one skilled in the art to practice the invention. The
following Examples are offered for illustrative purposes only, and
are not intended to limit the scope of the present invention in any
way. Indeed, various modifications of the invention in addition to
those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); N (Normal); mol (moles); mmol (millimoles); .mu.mol
(micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg
(kilograms); .mu.g (micrograms); L (liters); ml (milliliters);
.mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); .degree. C. (degrees Centigrade); h
(hours); min (minutes); sec (seconds); msec (milliseconds); ADCC
(antibody-dependent cellular cytotoxicity)); BsAb (bispecific
antibody); C.sub.L (constant domain of light chain); C.sub.H
(constant domain of heavy chain); CMC (complement-mediated
cytotoxicity); Fab (antigen binding fragment); Fc (crystallized
fragment); Fv (variable fragment (V.sub.L+V.sub.H)); EGFR
(epidermal growth factor receptor); HC (heavy chain); IGFR
(insulin-like growth factor receptor); LC (light chain); scFv
(singlechain variable fragment (V.sub.L and V.sub.H tethered by an
amino acid linker); VEGF (vascular endothelial growth factor);
VEGFR2 (vascular endothelial growth factor receptor 2); V.sub.H
(variable heavy domain); V.sub.L (variable light domain).
EXAMPLES
The present invention is described in further detain in the
following examples which are not in any way intended to limit the
scope of the invention as claimed. The attached Figures are meant
to be considered as integral parts of the specification and
description of the invention. All references cited are herein
specifically incorporated by reference for all that is described
therein. The following examples are offered to illustrate, but not
to limit the claimed invention.
Example 1
Construction of Expression Vectors
This example illustrates the nucleic acid construct used to
transform host cells.
Generally, both the heavy and light chain DNA coding sequences were
cloned into an expression plasmid that contained separate promoter
elements for each of the sequences and antibiotic resistance for
selection of bacterial cells that contain the expression plasmid.
The vector constructs also encode the heat-stable enterotoxin II
(STII) secretion signal (Picken et al., 1983, Infect. Immun.
42:269-275, and Lee et al., 1983, Infect. Immun. 42:264-268) for
the export of the antibody polypeptides into the periplasmic space
of the bacterial cell. Transcription of each chain is controlled by
the phoA promoter (Kikuchi et al., 1981, Nucleic Acids Res.,
9:5671-5678) and translational control is provided by previously
described STII signal sequence variants of measured relative
translational strength, which contain silent codon changes in the
translation initiation region (TIR) (Simmons and Yansura, 1996,
Nature Biotechnol. 14:629-634 and Simmons et al., 2002, J. Immunol.
Methods, 263:133-147). A schematic drawing of the knob and hole
plasmids is shown in FIGS. 2A and 2B, respectively.
While the present invention does not rely on specific antibody
binding sequences, and is applicable to any half-antibody
combinations, the Examples herein are directed to heteromultimeric
antibodies directed to c-met, EGFR, IL-4 and IL-13. Examples of
anti-c-met antibodies are given in U.S. Pat. No. 7,472,724, and
U.S. Pat. No. 7,498,420. Examples of anti-EGFR antibodies are given
in US Provisional Application 61/210,562 (filed 20 Mar. 2009), US
Pat. Appln. Pub. No. 20080274114 (published 6 Nov. 2008) and U.S.
Pat. No. 5,844,093 (granted 1 Dec. 1998). Examples of anti-IL-13
antibodies are described in U.S. Pat. No. 7,501,121 (granted 10
Mar. 2009), U.S. Pat. No. 7,615,213 (granted 10 Nov. 2009), WO
2006/085938 (published 17 Aug. 2006), US Pat Appln. Pub. No.
20090214523 (published 27 Aug. 2009), and U.S. Pat. No. 7,674,459
(granted 9 Mar. 2010). Examples of anti-IL-4 antibodies are
described in US Pat. Appln. Pub. No. US 20080241160 (published 2
Oct. 2008), and U.S. Pat. No. 6,358,509 (granted 19 Mar. 2002).
Each half-antibody had either a knob (protuberance) or a hole
(cavity) engineered into the heavy chain as described in U.S. Pat.
No. 7,642,228. Briefly, a C.sub.H3 knob mutant was generated first.
A library of C.sub.H3 hole mutants was then created by randomizing
residues 366, 368 and 407 that are in proximity to the knob on the
partner C.sub.H3 domain. In the following examples, the knob
mutation is T366W, and the hole has mutations T366S, L368A and
Y407V in an IgG1 backbone. Equivalent mutations in other
immunoglobulin isotypes is easily determined by one skilled in the
art. Further, the skilled artisan will readily appreciate that it
is preferred that the two half-antibodies used for the bispecific
be the same isotype. Half-antibodies of different isotypes may be
used but may need further mutations.
Although the vector described in this Example is for either the
anti-c-Met or anti-EGFR half-antibody, one skilled in the art will
readily appreciate that any antibody can be encoded in the plasmid.
The starting plasmid for all constructs used herein is the
previously described anti-tissue factor separate cistron plasmid,
paTF50, with relative TIRs of 1 for heavy and 1 for light (Simmons
et al., 2002, J. Immunol Methods, 263:133-147, and U.S. Pat. No.
6,979,556). An increase in the relative TIR strengths was used to
increase the expression titers of these half-antibodies.
Example 2
Heteromultimeric Protein Production Using Separate Cell
Cultures
The following example shows the production of heteromultimeric
proteins when the cells expressing the monomeric components are
grown in separate cultures. In this method the cells are grown and
induced to express the half-antibody in separate cultures. In one
method, the host cell cultures may be combined before protein
purification. In another method the components may be purified
first and then combined to form the heteromultimeric protein.
In both methods, a nucleic acid encoding the first hinge-containing
polypeptide (e.g., a half-antibody (knob)) is introduced into a
first host cell and a nucleic acid encoding the second
hinge-containing polypeptide (e.g., a half-antibody (hole)) is
introduced into a second host cell. Although this example
illustrates the formation of a BsAb one skilled in the art will
readily appreciate that the methods described are applicable to any
heteromultimeric protein comprising a hinge region, e.g.,
affibodies, etc.
Method #1--Independent Production of Knob Half-Antibody and Hole
Half-Antibody in Separate Cultures, Separate Purification of the
Half-Antibodies, Mixing and Redox to Form Intact BsAb.
Half-antibodies containing either the knob or hole mutations were
generated in separate cultures by expressing the heavy and light
chains using the constructs described in Example 1 in a bacterial
host cell, e.g., E. coli. See FIGS. 3B and 4A. In this Method #1
the knob half-antibody was an anti-EGFR and the hole half-antibody
was an anti-c-met. The expression plasmids of Example 1 were
introduced into E. coli host strains 33D3 (Ridgway et al. (1999) 59
(11): 2718) or 64B4 (W3110 .DELTA.fhuA .DELTA.phoA ilvG+.DELTA.prc
spr43H1 .DELTA.degP .DELTA.manA lacI.sup.q .DELTA.ompT) and
transformants were selected on carbenicillin containing LB plates.
Transformants were then used to inoculate an LB starter culture
containing carbenicillin, and this was grown overnight with shaking
at 30.degree. C. The starter culture was diluted 100.times. into a
phosphate limiting media C.R.A.P. (Simmons et al., 2002, J.
Immunol. Methods, 263:133-147) containing carbenicillin, and this
was grown for 24 hours with shaking at 30.degree. C. The cultures
were centrifuged, and the cell pellets frozen until the start of
antibody purification. The pellets were thawed and resuspended in
an extraction buffer containing 25 mM Tris-base adjusted to pH 7.5
with hydrochloric acid, 125 mM NaCl and 5 mM EDTA (TEB or Tris
Extraction Buffer) with a volume to weight ratio of 100 mL TEB per
5 grams of cell pellet, and extracted by disrupting the cells using
microfluidics by passing the resuspended mixture through a
Microfluidics Corporation model 110F microfluidizer (Newton, Mass.)
three times. The bacterial cell extract was then clarified by
centrifugation for 20 minutes at 15,000.times.g and the supernatant
collected and filtered through a 0.22 micron acetate filter prior
to purification.
Each half-antibody was purified separately by Protein A capture
followed by cation exchange chromatography. Clarified cell extracts
from the knob half-antibody were loaded onto a 1 mL HiTrap
MabSelect SURE column from GE Healthcare (Pistcataway, N.J.) at 2
mL/min. After loading the column was washed with 10 column volumes
(CV) of 40 mM sodium citrate, pH 6.0, 125 mM sodium chloride, and 5
mM EDTA followed by 5 column volumes of 20 mM sodium citrate at pH
6.0 to facilitate capture by the cation exchange column. The
affinity captured half-antibodies were eluted with 10 column
volumes (CV) of 0.2 mM acetic acid (pH 2-3) and directly captured
on a 1 mL HiTrap SP-HP strong cation exchange column from GE
Healthcare. The column was washed with 10 CV of buffer A containing
25 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8. The
half-antibodies were eluted with a linear gradient of 0-50% buffer
B (25 mM MES, pH 5.8 and 1 M sodium chloride (NaCl)). Both proteins
eluted between 20-40% B and the eluant peak as determined by UV
absorbance at 280 nm and by non-reducing SDS-PAGE analysis of the
collected fractions were pooled separately as the knob or hole
half-antibody. Both proteins generally exhibited a major elution
peak and all fractions that contained heavy chain and light chain
species that were oxidized to one another were included in the
pool. Analysis of the purified half-antibodies by reducing and
non-reducing SDS-PAGE are shown in FIG. 4B. The results indicate
that most of the expressed and captured protein is 75 kD in size.
We confirmed this by ESI-TOF mass spectrometry shown in FIG. 4C.
The mass of the half-antibodies were the expected masses indicating
that there were no disulfide adducts on any cysteine, including the
two cysteine residues in the hinge region. To determine if the
hinge cysteines were reduced exhibiting a reactive free thiol, the
proteins were reacted in at a neutral pH with 1 mM N-ethylmaleimide
(NEM) for one hour before analysis by mass spectrometry. The mass
of the protein was unchanged indicating that the hinge cysteines
were oxidized to each other most likely in an intrachain disulfide,
e.g., a cyclic disulfide. In order to assemble a fully intact,
bispecific antibody using these two half-antibodies (knob and
hole), it was necessary to first reduce the intrachain disulfides
at the hinge region to liberate the cysteine free thiols so that
they could subsequently be oxidized to the other heavy chain to
form the 150 kD bispecific antibody.
To accomplish the annealing, reduction and reoxidation of the two
complementary half-antibodies to form the intact bispecific
molecules the following procedure was developed. After independent
isolation, the purified proteins were combined together at equal
mass in the Pool step of the procedure (shown in FIG. 5A), the pH
of the pool was adjusted to 7.5 by adding one-tenth volume of 1 M
Tris, pH 7.5, and proteins were reduced with 0.5 mM
Tris[2-carboxyethyl] phosphine (TCEP) at room temperature. After
reduction for 2 hours the pooled proteins were buffer exchanged
into 25 mM Tris, pH 7.5, and 125 mM NaCl using 5 mL Zeba Desalt
spin columns (Pierce, Rockford, Ill.) resulting in a volume of
about 4 mLs of a protein concentration of 1 mg/mL. The proteins
were then annealed by heating the mixture to 52.degree. C. for 25
minutes followed by cooling to room temperature, about 20.degree.
C. The annealed antibodies were concentrated using 10 kD MW cutoff
spin concentrators to a volume of 0.5 mL with a protein
concentration of about 8 mg/mL and oxidized by the addition of 300
micromolar dehydroascorbic acid (DHAA) to the reaction mixture from
a stock solution of 100 mM DHAA dissolved in dimethylsulfoxide. The
amount of DHAA added for oxidation is about 10-fold excess over the
protein molar concentration. After oxidation overnight at room
temperature, the oxidized material was run on an S-200 gel
filtration column (22 mL S200 Tricorn from GE Healthcare) in a
buffer containing 25 mM MES pH 6.0 and 300 mM NaCl. The intact
antibody was pooled and diluted 10-fold in water. The BsAb protein
was then purified by weak cation exchange chromatography using a
carboxymethyl (CM) resin (1 mL HiTrap CM-FF, GE Healthcare) with a
pH gradient elution from 4.5 to 9.2. The buffer A and B composition
consisted of 20 mM sodium citrate, 30 mM MES, 20 mM HEPES, 20 mM
imidizole, 20 mM Tris, 20 mM CAPS, and 25 mM NaCl, where the A
buffer is adjusted to pH 4.2 with HCl and the B buffer is adjusted
to pH 9.2 (or 10.4) using NaOH. The purified material obtained
after CM chromatography was analyzed by mass spectrometry to
determine the exact molecular composition (FIG. 4D). Mass spec
analysis indicated that the only detectable intact antibody product
was with a MW of 146,051.89, which matches nearly identically with
the heterodimeric knob-hole species anti-EGFR/anti-c-met with a
theoretical MW of 145,051.75. The yield of this procedure,
beginning with about 2 mg of the knob and 2 mg of the hole was
about 0.5-1 mg.
For large scale production of antibodies for in vivo
experimentation such as the determination of pharmacokinetic
properties in non-human primates, 100 mg to gram scale quantities
of antibody are needed. We developed a procedure using a separate,
independent culture for each half-antibody as shown in FIG. 5A to
produce intact bispecific antibodies in these quantities. For these
preparations, 10 liter fermentations were required to produce cell
pellets or whole broth with sufficient quantities of antibody
(Simmons et al., 2002. J. Immunol. Methods, 263:133-147, and U.S.
Pat. No. 6,979,556). In the course of experimentation either cell
pellets or bacterial whole broth were used for biomass containing
expressed half-antibodies. In some cases, a significant fraction of
the antibody had leaked out into the media, where whole broth gave
higher yields. For cell pellets, the material was resuspended in
extraction buffer containing 25 mM Tris, pH 7.5, 5 mM EDTA, and 125
mM NaCl and lysed by microfluidization using a Model HC80003A
microfluidizer from Microfluidics (Newton, Mass.). Whole broth was
directly microfluidized without the addition of additives. In both
cases, three passes of the material through the instrument was
done. In this example, we prepared 500 mg of two versions of a
bispecific antibody targeting the cytokines interlukin-4 (knob) and
interleuikin-13 (hole).
The first version of the bispecific contained a human IgG1a Fc with
only the knob and hole mutations and the second contained a further
modified Fc with two mutations, T307Q and N434A, that lead to a
greater affinity for the neonatal Fc receptor (FcRn). The second
versions are expected to impart a slower clearance and longer
half-life for the antibody. The hole antibody (targeting IL-4) and
the knob antibody (targeting IL-13) of both versions of the Fc
(WT-Fc for the former and FcRn-variant for the later) were both
grown separately in 10 liter fermentation and the whole broth
containing growth media and bacterial cells were homogenized and
purified independently. After microfluidization of the whole broth,
the extract was treated with an equal volume of 0.4%
polyethyleneimine (PEI) (pH 9.0) to prepare the extract for
clarification by centrifugation. The mixture was stirred for 3
hours at room temperature or overnight at 4.degree. C. PEI caused
extensive precipitation of the extract which was clarified by
centrifugation at 15,000.times.g for 45 minutes. The supernatant
was subsequently filtered by 0.22 micron filters before loading on
a 100 mL Mab Select SURE Protein A capture column. The extract was
loaded at 20 ml/min and washed with 40 mM sodium citrate, pH 6.0,
and 100 mM NaCl until the UV absorbance at 280 reached a stable
baseline, generally about 10 column volumes (CV). The wash buffer
was changed to 20 mM sodium citrate, pH 6.0 and washed for about 2
CV. The captured half-antibody was eluted using 0.2 M acetic acid.
After isolation by Protein A the antibodies were purified by cation
exchange chromatography using S-FF resin (GE Healthcare) or gel
filtration chromatography using S200 resin (GE Healthcare) to
remove impurities and aggregates. The purified half-antibodies were
mostly the .about.75 kD species as seen in FIG. 5B. After the
second isolation step, 500 mg of each half-antibody were pooled
together at a concentration of 1 mg/mL and the pH was adjusted to
7.5 using 1 M Tris, pH 7.5. The mixture was heated to 37.degree. C.
in an incubator and monitored by gel filtration for the emergence
of the 150 kD antibody species. After 2 hours, the annealing was
complete showing complete conversion to the dimeric 150 kD species
and the mixture was cooled to room temperature. The proteins were
reduced by the addition of 2 mM DTT for two hours at 24.degree. C.
and subsequently concentrated to 20 mg/mL using 10 kD cutoff spin
filters. The concentrated solution was oxidized by dialysis
overnight in a buffer containing only 25 mM Tris, pH 8.0. The
oxidized material was subsequently analyzed for purity and
aggregation. The intact antibody species was determined by mass
spectrometry to be the intact, fully oxidized heterodimeric
bispecific molecule however gel filtration and SDS-PAGE analysis
indicated the presence of significant amounts of aggregate, some of
which was clearly the result of disulfide linked multimers (DATA
not shown). To further purify the bispecific antibody for in vivo
experimentation, the antibody was separated over an S-200 gel
filtration column in Tris, pH 7.5 and 125 mM NaCl. The purified
material exhibited a greater than 30% loss of material due to the
removal of introduced aggregates. For the final stages of the
preparation, the protein was adhered to a cation exchange column,
washed with 0.1% TX114 in 50 mM sodium acetate, pH 5.0, to remove
contaminating endotoxin, and eluted with a high pH buffer
containing 50 mM Tris, pH 8.0. The eluted protein was then
formulated by dialysis into a buffer suitable for in vivo
experimentation and stored at 4.degree. C. The final material
consisting of the WT-Fc and the FcRn-variant was analyzed by
SDS-PAGE, mass spectrometry, LAL assays for determining
contaminating endotoxin levels, and gel filtration analysis. The
results of the SDS-PAGE are shown in FIG. 5C, and indicate that the
major species is the intact bispecific antibody at 150 kD. FIG. 6A
shows the biological activity of the antibodies in a TF-2 cell
proliferation assay testing neutralization of the cytokines IL-4
and IL-13. For the assay, anti-IL-4/IL-13 bispecific, anti-IL-4 and
anti-IL-13 antibodies were used at a starting concentration of 25
ug/ml and serially diluted 10 fold in a 96 well culture plate
(Falcon, Cat#353072) to a final concentration of 0.025 pg/ml in
assay media (culture media without rhGM-CSF) or assay media
containing 0.4 ng/ml human IL-4 (R&D Systems, Catalog #204-IL)
plus 20 ng/ml human IL-13 (Genentech Inc.) in a final volume of 50
ul/well. Diluted antibodies were pre-incubated for 30 minutes at
37.degree. C.
Following preincubation, TF-1 cells cultured in RPMI 1640
(Genentech, Inc.) 10% Fetal Bovine Serum (HyClone, Cat#
SH300071.03), 2 mM L-glutamine 100 units/mL Penicillin 100 .mu.g/mL
Streptomycin (Gibco, Cat#10378) and 2 ng/mL rhGM-CSF (R&D
Systems, Cat #215-GM) were washed 2 times with assay media and
resuspended in assay media to obtain a final concentration of
2.times.10.sup.5 cells/ml. 50 ul of cells were added to each well
containing either the diluted antibodies, assay media plus IL-4 and
IL-13 cytokines (maximal proliferation control) or assay media
alone (background control). All samples were plated in duplicate.
Plates were incubated at 37.degree. C. at 5% CO.sub.2 for 4 days. 1
uCi .sup.3H Thymidine (Perkin Elmer, Cat# NET027005MC) was added to
each well during the final 4 hrs of incubation. Plates were
harvested onto a Unifilter-96 GF/C (Perkin Elmer, Cat#6005174)
using a Packard Filtermate, .sup.3H thymidine incorporation was
measured using a TopCount NXT (Perkin Elmer). Data was plotted
using KaleidaGraph. The results indicate that the WT
anti-II-4/anti-IL-13 bispecific antibody is as effective as IgG
antibody combinations of IL-4 and IL-13 in neutralizing IL-4 and
IL-13 activity.
The two antibodies (WT anti-IL-4/anti-IL-13 and FcRn-variant
anti-IL-4/anti-IL-13) were then tested for their pharmacokinetic
(PK) properties in cynomologous monkey. Using a single dose
injection, the WT molecule formulated in 20 mM histidine-acetate,
pH 5.5, 240 mM sucrose, and 0.02% Tween 20 at 10.8 mg/mL and 1
mg/mL and the FcRn-variant in 20 mM sodium phosphate, pH 7.5, 240
mM sucrose, and 0.02% Tween 20 at 10.5 mg/mL, were administered by
IV injection. The dosing level was 20 mg/kg and 2 mg/kg for the two
WT concentrations and 20 mg/kg for the FcRn-variant. Serum samples
from two female and two male monkeys that were injected with the
three treatments were taken periodically over the course of 42
days. The serum samples were assayed for the intact bispecific
antibody by ELISA wherein one antigen, either IL-4 or IL-13, was
coated onto the plates and the antibody subsequently captured from
the serum. The amount of captured bispecific antibody present was
determined by detection with a second biotinylated ligand either
IL-13 or IL-4 (whichever ligand had not been coated onto the
plates), and enzyme-coupled streptavidin. The results in FIG. 6B
shows the expected two compartment clearances of the three samples.
The PK properties of the two different versions of the antibody are
shown in Table 2 in comparison to two other antibodies that are
derived from CHO production hosts (Avastin and Herceptin) and
contain Fc-glycosylation. It is clear that the E. coli produced
bispecific antibody is similar to the CHO derived antibodies from a
standard process and that the FcRn-variant has a longer
half-life.
TABLE-US-00002 TABLE 2 Population Mean Vc CL T1/2 (% RSE) (mL/kg)
(mL/kg/day) (day) WT 29.0 (9.48) 4.49 (7.66) ~10 FcRn 15.8 (5.72)
2.11 (2.47) ~18 Avastin 4.3 ~12 Herceptin 5.5 ~9
Method #2--Production of Knob Half-Antibody and Hole Half-Antibody
in Separate (i.e., Independent) Cultures, Mixing Whole Broth Prior
to Purification of the Half-Antibodies and Lysis without the
Addition of a Reductant to Form Intact BsAb.
This method was an attempt to reduce the number of steps in the
process by purifying the knob and the hole half-antibodies at the
same time. Therefore, fermentation broths were mixed prior to
pelleting and resuspending in extraction buffer. It was thought
that each host cell would release its expressed half-antibody
containing the cyclic disulfide within the hinge region into the
extraction buffer upon cell membrane disruption. Subsequently, the
purification of both half-antibodies could be done simultaneously
followed by the redox-annealing step to form the intact BsAb.
Surprisingly, we discovered that the knob-hole antibodies
heterodimerized and oxidized on their own to form a full length
antibody (.about.150 kD) at greater than 20% of the combined total
of the intact and half-antibody (.about.75 kD) (see Table 3).
The knob half-antibody and hole half-antibody expressing host cells
were grown and induced in separate cultures using the process as
described in Method #1, supra. The whole cell fermentation broth
from each culture was mixed with the other at three different
volume ratios and then centrifuged to form a single cell pellet.
The whole cell fermentation broths were mixed together to a final
volume of 500 mL at an (anti-c-met):(anti-EGFR) ratio of 1:1, 2:1
or 1:2, with the intent to match recovery of the two antibodies in
relatively equal abundance and knowing that the anti-EGFR half
antibody expressed similar to the cMet antibody under the same
conditions. Each cell pellet was resuspended in extraction buffer
and lysed. Protein was extracted and purified by Protein-A
chromatography followed by cation exchange chromatography as
described in Example 2, Method #1. The extraction buffer contained
25 mM Tris, pH 7.5, 125 mM NaCl, and 5 mM EDTA. When purified
separately, each of the knob-half-antibody and hole-half-antibody
form a cyclic disulfide within the hinge region, i.e., an
intrachain disulfide, preventing covalent association of the knob
and hole heavy chains. However, it was found that when the first
and second host cells were lysed together either after co-culturing
or after mixing whole fermentation broths prior to centrifugation,
there was some level of assembly into the intact antibody species.
FIG. 7 shows the intact antibody species observed in the three
ratios. This suggested that modifications to the procedure could
result in spontaneous formation of the intact bispecific antibody
which could substantially eliminate the need for additional
chemistry steps.
Quantitation of the two protein species was done by separating 5
micrograms of protein by SDS-PAGE using a Novex 4-20% Tris-Glycine
gel (Invitrogen, Carlsbad Calif.). After electrophoresis the gel
was stained with colloidal Coomassie stain containing 150 mM
ammonium sulfate, 1.74 M acetic acid, 10% methanol and 0.4 g/L
Coomassie Dye R250 in water. The gel was destained with 10% acetic
acid in water and subsequently equilibrated in Gel-Dry Drying
Solution (Invitrogen) and dried between two sheets of cellophane.
After drying the gel, the protein bands were quantified by the
Odyssey IR imaging system (LI-COR Biosciences, Lincoln, Nebr.) at
700 nm.
TABLE-US-00003 TABLE 3 Licore fluorescent signals for intact
antibodies and half-antibodies after mixed isolations from two
separately grown knob and hole cultures. [this is a measure of a
hinge] Volume Ratio Combined % of 150 (c-met:EGFR) 150 kD RFUs 75
kD RFUs RFUs RFU/total 1:1 36.01 98.78 134.8 26.72 2:1 36.8 107
143.8 25.59 1:2 34.64 107.83 142.5 24.31
Method #3--Production of Knob Half-Antibody and Hole Half-Antibody
in Independent Cultures, Independent Centrifugation, Pellets Mixed
& Resuspended Followed by Lysis, and Purification of the BsAb
without the Addition of a Reductant.
This method is an attempt to reduce the number of steps in the
process by purifying the knob and the hole at the same time.
The cells are cultured independently and pelleted by
centrifugation. The pellets are mixed and resuspended together in
extraction buffer. It is believed that the half-antibodies will be
released into the extraction buffer upon disruption of the cell
membranes and that a similar product profile will be seen as with
Method #2, above.
Example 3
Heteromultimeric Protein Production Using a Single Mixed Cell
Culture
This example illustrates the formation of heteromultimeric proteins
from a culture comprising two host cell populations, wherein there
is no addition of a reductant in the process.
Method #4--Production of Knob Half-Antibody and Hole Half-Antibody
from Different Cell Populations in the Same Culture to Form Intact
BsAb without the Addition of Reductant.
Co-culture experiments were first performed in 0.5 liter shake
flasks with two different E. coli transformants containing either a
knob or hole half-antibody. For this experiment, a starter culture
of both the knob (anti-EGFR) and hole (anti-cMet) half-antibodies
were produced by overnight culture in LB-media (100 .mu.g/ml
carbenicillin) in 5 mL cultures at 30.degree. C. The overnight
cultures of equal OD.sub.600 were used to inoculate 500 ml complete
CRAP-media (100 .mu.g/ml carbenicillin) in three different ratios
(anti-EGFR:anti-cMet; 1.5:1, 1:1 and 1:1.5) keeping the total seed
volume to 1/100 of the culture. Cells were grown for 24 hrs at
30.degree. C., 200 rpm. The cells were then pelleted by
centrifugation (6750.times.g, 10 minutes, 4.degree. C.) and used
for purification.
The cells were resuspended in extraction buffer containing 25 mM
Tris, pH 7.5, 5 mM EDTA, and 125 mM NaCl at a ratio of 100 mL per
10 g cell pellet. After extraction by microfluidization and
preparation for chromatography as described in Example 2, the cell
extracts of the three different ratios were purified by first
capturing the bispecific antibody on a Mab Select SURE 1 mL HiTrap
column (GE Healthcare, S. San Francisco, Calif.) and with a column
wash buffer containing only 40 mM sodium citrate at pH 6.0. After
washing and elution as described in Example 2, the protein A
capture pools were loaded onto an SP-HP cation exchange column and
purified as described in Example 2. After separation by cation
exchange, the chromatographic peaks from each of the three
purifications were pooled and concentrated to a volume of about
50-100 microliters, and with a protein concentration of about 15
mg/mL. The initial inoculation ratios appeared to make a difference
in the final amount of intact antibody, and this was a higher
proportion of intact bispecific antibody to lower molecular weight
forms than was observed when the cell pellets were mixed together
after overnight culturing at 37.degree. C. See Table 4.
TABLE-US-00004 TABLE 4 Inoculation 150 kD 75 kD Combined % of 150
RFU/ Ratio RFUs RFUs RFUs total 1.5 to 1 11.71 10.28 22.0 53.25 1
to 1 9.09 8.96 18.1 50.36 1 to 1.5 7.28 8.71 16.0 45.53
To determine if co-culture can be extended to the 10 liter
fermentation scale, which is critical for scale up procedures,
several experiments were done with the anti-EGFR and anti-cMet
half-antibodies. For 10 liter fermentations, an inoculation
starting culture was used that contained a 1:1 cell ratio of
anti-EGFR and anti-cMet. The 10 liter co-cultures were grown under
identical conditions as for the single half-antibody cultures
described in Example 2. Either cell pellet or whole broth was used
for extraction and isolation of the antibody material, also as
described above. For extraction of material from the cell pellets,
about 2.5 kg of paste was produced from one 10 liter fermentation.
The cell pellets were resuspended in 5 Liters of buffer containing
25 mM Tris, pH 7.5, and 125 mM NaCl. The pellet was treated with a
polytron mixer for 2 minutes prior to resuspending the pellet, and
then microfluidized, clarified, and prepared for Protein A capture
as described in Example 2. The fermentation experiment was repeated
two more times and the results of the co-culture isolation from 10
liter fermentors are shown in FIG. 8C. Mass spectrometry was used
to characterize the .about.150 kD protein and the .about.75 kD
protein to determine the molecular components. To our surprise, the
dominant upper MW protein is the bispecific antibody and the
.about.75 kD protein was primarily the cMet half-antibody due to
its differential expression profile. This indicates that the
bispecific antibody has completely formed without the need for
additional chemistry steps. Because the bispecific antibody is a
1:1 stoichiometric combination of the knob and hole
half-antibodies, the presence of only a 75 kD protein indicates
that the majority of the limiting half-antibody had been
spontaneously incorporated into the intact bispecific antibody.
This observation led to the development of a simplified expression
and purification scheme as shown in FIG. 8D. After protein A
capture, the antibody was diluted 1:1 with a buffer containing 1.5
M ammonium sulfate and 25 mM sodium phosphate pH 6.5 and loaded
onto a hydrophobic interaction column (HIC) Dionex Pro Pac HIC-10
4.6 mm.times.100 mm (Sunnyvale, Calif.). A gradient of 30-60% B,
with the A buffer composed of 25 mM sodium phosphate, pH 6.95, and
1.5 M ammonium sulfate, and the B buffer composed of 25 mM sodium
phosphate, pH 6.95, and 25 isopropyl alcohol. Proteins were
separated with a 15 CV gradient. The protein separated into two
major species, one containing the intact bispecific antibody and
the other containing the excess anti-EGFR half-antibody. The
results of the chromatographic separation are shown in FIG. 8E. The
fractions containing the intact antibody were pooled and treated to
remove any remaining contaminating endotoxin by adherence to an
S-FF column in a 25 mM sodium acetate buffer at pH 5.0, washing
with the same acetate buffer containing 0.1% Triton X114, and then
removing the detergent by washing with the starting acetate buffer.
The protein was eluted from the S-FF column using 25 mM Tris, pH
8.0, pooled, and analyzed by SDS-PAGE, mass spectrometry and LAL
assays for endotoxin. The protein contained 0.076 EU/mg of
endotoxin in the final preparation, indicating that it is suitable
for in vivo applications. The final characterization is shown in
FIG. 8F. The SDS-PAGE analysis shows a majority of the protein to
be the final intact bispecific antibody, and the mass spec analysis
shows the expected molecular weight for the bispecific antibody,
and the lack of any contaminating species, in particular the
homodimeric forms that could be present. The comparison of the
modified procedure using coculturing compared to the procedure that
requires annealing and redox chemistry is shown in FIG. 8G.
Method #5--Production of Knob Half-Antibody and Hole Half-Antibody
in the Same Culture to Form Intact BsAb Using Differing Knob:Hole
Ratios.
This example shows that host cells using similar expression
constructs (differing only in the half-antibody to be expressed) do
not outgrow each other and produce intact BsAb.
Experiments have demonstrated that controlling the ratio of either
chain is easily done by adjusting the inoculation ratio prior to
expansion and expression. The two strains do not outgrow one
another.
To determine if the ratio of inoculation is preserved over the
fermentation of a co-culture, an experiment was conducted to
determine the amount of the knob or hole heavy chain that was
present at the end of a 24 hour fermentation of co-cultures with
different cell ratios. Cells harboring either the knob (anti-EGFR)
or hole (anti-c-Met) plasmid were grown separately in LB-media (100
.mu.g/ml carbenicillin) over night at 30.degree. C. The starter
culture was used to inoculate complete CRAP-media (100 .mu.g/ml
carbenicillin) with different ratios of overnight culture keeping
the combined inoculation volume at 1:100 of the final culture. The
ratios tested for anti-EGFR:anti-c-Met were 10:1, 5:1, 2:1, 1:1,
1:2, 1:5, and 1:10. After culturing for 24 hrs at 30.degree. C.
cell samples were obtained and analyzed by non-reduced SDS-PAGE
(12% TrisGlycine) followed by Western blotting with Goat anti-Human
IgG-Fc Antibody HRP conjugated (Bethyl Laboratories, Inc.,
Montgomery, Tex.). The heavy chains of the two species resolve by
SDS-PAGE and the result is shown in FIG. 8B. The amount of each
half-antibody correlates with the inoculation ratio of the
co-culture, indicating that cells harboring plasmids encoding
different half-antibodies do not outgrow each other in a
co-culture.
Method #6--Production of Knob Half-Antibody and Hole Half-Antibody
in the Same Culture to Form Intact BsAb--Membrane
Permeabilization.
This example shows that membrane permeabilization releases the
half-antibodies into the media and with the subsequent formation of
an intact BsAb without the need for additional chemistry (e.g.,
redox or coupling).
It is known that mutations leading to the loss of lipoprotein
synthesis alters the cell membrane of E. coli conferring leakiness
of periplasmic proteins into the media and also renders E. coli
hypersensitive to EDTA (Hirota, Y. et al. PNAS 74:1417-1420
(1977)). The release of expressed antibody from strain 65G4 (W3110
.DELTA.fhuA .DELTA.phoA ilvG+.DELTA.prc spr43H1 .DELTA.degP
.DELTA.manA lacl.sup.q .DELTA.ompT .DELTA.lpp) with and without
addition of EDTA was compared. Cells expressing either a-IL-4
(hole) or .alpha.-IL-13 (knob) were co-cultured as described in
Method #4 in a 1:1 ratio and grown in an incubator shaker at 200
rpm for 20 hrs at 30.degree. C. At the end of the incubation the
culture was split into three equal aliquots. One sample served as a
control with no EDTA added. To the other two samples EDTA, pH 8.0,
was added to 10 mM final concentration. Incubation was continued
for all samples for 30 minutes, after which one of the EDTA treated
samples had MgCl.sub.2 added to 20 mM final concentration. All
samples were incubated for an additional 30 minutes in the
incubator shaker before removing cells by centrifugation
(9200.times.g, 20 minutes, 4.degree. C.) and the supernatant
filtered through a GF/F filter (Whatman, Piscataway, N.J.) and 0.2
.mu.m PES filter (Nalgene, Rochester, N.Y.). DNasel, bovine
pancreas (Sigma, St. Louis, Mo.) can be added to 4 mg/l to improve
filtration.
The filtered supernatant was then directly loaded over a 1 mL
Protein A MabSelect SURE HiTrap column (GE Healthcare) as described
previously. The captured protein was eluted with acetic acid as
described above and the peak recovery of the protein can be seen in
FIG. 9A. The results show that the total UV absorbance increases in
the EDTA treated samples. This absorbance is intact bispecific
antibody and excess half-antibody. See FIGS. 9B and 9C.
In a separate experiment the anti-IL-4 and anti-IL-13
half-antibodies were expressed separately or as a 1:1 co-culture of
65G4 cells. Cells were cultured as described above (Method #4) with
the exception of supplementing the complete CRAP media with
Silicone Antifoam (Fluka, Buchs, Switzerland) to 0.02% (v/v). After
culturing the cells for 24 hrs, 30.degree. C., 200 rpm in an
incubator shaker, EDTA, pH 8.0, was added to 10 mM final
concentration and incubation continued for one hour before adding
MgCl.sub.2 to 20 mM. Cells were harvested by centrifugation
(6750.times.g, 10 minutes, 4.degree. C.), the supernatant filtered
(0.2.mu.PES, Nalgene, Rochester, N.Y.) and antibodies were captured
by protein A as described above and analyzed by SDS-PAGE and mass
spectrometry. The results shown in FIG. 9D indicate that intact
bispecific antibody formation is observed only in the presence of
both halves of the bispecific. Additionally, the majority of the
anti-IL-13 antibody was incorporated into the bispecific antibody
without any additional redox chemistry as mass spec analysis
indicated that the 75 kD protein band was mostly the anti-IL-4
half-antibody. The protein A purified bispecific antibody was
diluted 1:1 with ammonium sulfate buffer and further purified with
a 7.5 mm.times.150 mm ProPac HIC-10 column (Dionex, Sunnyvale,
Calif.) using the same procedure as described in Example 3. The
intact bispecific antibody was found to elute at a retention time
of 99.68. This peak was pooled and analyzed by SDS-PAGE in
non-reducing conditions and found to be nearly entirely composed of
the intact antibody species. To confirm that this protein was a
pure heterodimeric bispecific molecule, we analyzed the protein by
ESI-TOF LC/MS. About 10 micrograms of the bispecific antibody were
injected onto a PLRP-S 300 A 3 micrometer 50.times.2.1 mm reverse
phase column (Polymer Laboratories) and separated by a 4.3 minute
gradient of 34-45% 0.05% TFA and acetonitrile using an Agilent 1200
Series HPLC and a flow rate of 0.5 mL/min and a column heater at
80.degree. C. Protein eluting from the LC was analyzed by an
Agilent 6210 TOF. A single peak containing protein was observed,
and this peak was deconvoluted using Agilent Mass Hunter software
version B.02.00 using a mass range of 50,000-160,000, 1.0 Da step,
30.0 S/N threshold, average mass of 90, an unlimited mass range and
an isotope width set to automatic. The majority of the signal
representing the expected mass of the bispecific molecule. The mass
for the intact heterodimeric bispecific calculated from the amino
acid sequence is 144,044 which is within 1-2 Daltons of the
measured mass, whereas the calculated masses of the possible
homodimeric proteins are 144,954.6 for anti-IL-4 and 145, 133.4 for
anti-IL-13.
We tested if different inoculation ratios would again persist
throughout the culture for this set of antibodies and also in the
context of the Ipp deletion of 65G4. Seed cultures with either
anti-IL-4 (hole) or anti-IL-13 (knob) of equal OD.sub.600 were used
to inoculate 500 ml CRAP media at 2:1 and 1:2 ratios, cultured and
permeabilized at the end of the fermentation as described before
(see Method #6). The two different media preparations were purified
by Protein A capture followed by HIC separation as described above,
except that the pH of the HIC A and B buffers were lowered to 6.5.
The results of the two different starting culture ratios are shown
in FIG. 9E. It is observed that the majority of the protein is the
intact bispecific antibody. The other peaks were characterized by
mass spectrometry and labeled on the FIG. 9E. The anti-IL-13
half-antibody is slightly detected, and a significant amount more
of anti-IL-4 is seen. In the 33/66 ratio of anti-IL-4 to
anti-IL-13, there is more anti-IL-13 observed with a slight amount
of anti-IL-4 remaining. Here we see that the ratio of inoculation
is maintained throughout the culture and that the optimization of
the process could be achieved by balancing the ratios of expressed
antibody halves through manipulating the started culture
ratios.
We have continued to test this process of co-culture expression in
delta-Ipp cells on a number of different antibody variants. We show
in FIG. 9F the final purified proteins after formulation post HIC
chromatography of a few exemplary half-antibodies.
Example 4
Heteromultimeric Protein Libraries
This example illustrates the construction of a heteromultimeric
protein library.
Certain methods that may be used to screen mixtures of bispecific
antibodies or to rapidly generate large arrays of bispecific
antibodies using the methods described.
Method #7
In some cases the choice of bispecific antibody is not known, but
could be the result of the combination of many different
half-antibodies. Alternatively, a specific target combination may
be desired, e.g., anti-IL-4/anti-IL-13 but there are a number of
candidate half-antibodies to choose from. Finding the specific
half-antibody combination that yields the best binding or efficacy
may be accomplished by combining the half-antibodies in a matrix
format, one can produce many bispecific antibody variants rapidly.
For this experiment, one antibody such as anti-CD3 can be produced
at about 10-fold (or greater) excess over the amount of antibody
needed for screening. This molecule can then be annealed and
oxidized using the procedure described in Example #1. About one
tenth of the total amount of the first antibody can be used to
combine with an equal amount of about 10 half-antibodies targeting
different antigens (such as anti-CD19, anti-CD20, etc.) as
diagramed in FIG. 10. If an additional primary half-antibody is
needed to combine with the second half-antibody repertoire, this
can be done to yield a set of screening molecules.
In a second modification of the method, the primary antibody (such
anti-CD3) can be grown as a co-culture using "normal" E. coli host
cells or with a mutant strain having a non-functional lipoprotein
phenotype. This half-antibody can then be systematically added to
each of the variable half-antibodies producing an array of
bispecific molecules all containing the primary targeting
half-antibody.
Method #8
The primary half-antibody can be combined with a host of
alternative partnering half-antibodies in a manner that consists of
producing this half-antibody in sufficient quantity to combine with
all of the other antibody half-antibodies combined. A bulk
annealing can then be performed in a single reaction such that the
primary half-antibody is either the knob or the hole version of the
heavy chain and the set of secondary targeting half-antibodies are
the complimentary mutant. Here, a complex mixture of antibodies can
be produced that may be useful treating disease as a
combination.
Alternatively, a co-culture approach using the methods described in
the above Examples can be used to produce a complex mixture of
bispecific antibodies with a set primary half-antibody and a
variable secondary half-antibody. Such a mixture could then be
isolated in bulk and used as a screening material such that a
positive result in the pool of bispecific variants could be later
deconvoluted to determine the active bispecific antibody species,
or the combined mixture could be used as a more effective
therapeutic mixture.
Example 5
In Vitro Activity
This example that the bispecific antibodies described herein
possess activity in in vitro systems. Two cell lines were employed
in this Example 5 and in Example 6, below. In these experiments
KP4, a pancreatic ductal carcinoma cell line, and A431, an
epidermoid carcinoma cell line, are both strongly driven by Met or
EGFR, respectively, therefore these are good cell lines and tumor
xenografts to explore efficacy of bsAb against each target
independently.
The KP4 cell line was obtained from the Riken BioResource Center
Cell Bank (Cell line #: RCB1005; 3-1-1 Koyadai, Tuskuba-shi,
Ibaraki 305-0074 Japan). The A431 cell line (CRL-1555) was obtained
from the American Type Culture Collection (ATCC, Manassas,
Va.).
Cancer cells, A431, were washed once with PBS, re-suspended in
serum-free medium, counted, and then added to 96-well plates (2500
cells/well). Cells were then treated with human HGF (0.5 nM) and
TGF.alpha. (0.05 nM) alone or with a dose range of either (1)
anti-EGFR, (2) Anti-c-met antibody ("one-armed" c-met), (3) the
combination of anti-EGFR and Anti-c-met antibody or (4) the
bispecific anti-EGFR/anti-c-met antibody. Three day AlamarBlue.TM.
assays were performed according to manufacturer's recommendations
(BioSource International; Camarillo, Calif.). IC.sub.50 values were
determined by nonlinear regression analysis with a four-parameter
model (KaleidaGraph ver. 3.6, Synergy Software; Reading, Pa.).
In the KP4 cell assay which is Met dependent in vitro and in vivo,
growth stimulated by treatment with TGF-alpha and HGF can be
inhibited by Anti-c-met antibody, the combination of Anti-c-met
antibody and anti-EGFR, and the bispecific antibody. Treatment with
anti-EGFR shows limited activity as a single agent in these cells.
There was, however, more potent inhibition by the bispecific
antibody in KP4 cells than anti-c-met alone or anti-c-met plus
anti-EGFR Abs added separately. In A431 cells, which are primarily
driven by EGFR, neither the anti-EGFR antibody nor the anti-c-met
antibody alone were able to significantly inhibit cell
proliferation. The combination of both molecules did show some
inhibition of cell proliferation, however, the bispecific antibody
exhibited greater activity at the same concentrations. Also, the
cells exhibited apoptosis in addition to anti-proliferation.
In these assays the bispecific antibody showed improved performance
relative to the other antibodies alone or the combination of
anti-Met and anti-EGFR antibodies added separately. These data
suggest that it is the arrangement of anti-Met and anti-EGFR
antibodies together on one antibody that makes the bispecific
superior. The results are shown in FIG. 11.
Example 6
In Vivo Activity
This example demonstrates that the bispecific antibodies described
herein possess activity in in vivo models.
Female nude mice that were 6-8 weeks old and weighed 22-30 g were
obtained from Charles River Laboratories, Inc. (Hollister, Calif.).
The mice were housed at Genentech in standard rodent micro-isolator
cages and were acclimated to study conditions for at least 3 days
before tumor cell implantation. Only animals that appeared to be
healthy and that were free of obvious abnormalities were used for
the study. All experimental procedures conformed to the guiding
principles of the American Physiology Society and were approved by
Genentech's Institutional Animal Care and Use Committee. Mice were
injected subcutaneously with either human KP4 pancreatic cancer
cells (5 million cells in Hank's Balanced Salt Solution (HBSS) plus
Matrigel (BD Biosciences) per mouse) or human A431 epidermoid
carcinoma cells (5 million cells in HBSS plus Matrigel/mouse). When
tumors reached .about.150 mm.sup.3, mice were randomized and
treated with vehicle or the bispecific EGFR/c-met (bsEGFR/c-met)
(50 mg/kg IP 1.times./week) for 2 weeks.
Tumor volumes were measured in two dimensions (length and width)
using Ultra Cal-IV calipers (Model 54-10-111; Fred V. Fowler Co.;
Newton, Mass.) and analyzed using Excel, version 11.2 (Microsoft
Corporation; Redmond Wash.). Tumor inhibition graphs were plotted
using KaleidaGraph, version 3.6 (Synergy Software; Reading, Pa.).
The tumor volume was calculated with the following formula: Tumor
size(mm.sup.3)=(longer measurement.times.shorter
measurement.sup.2).times.0.5
The data was analyzed by the mixed modeling approach described
below. Here, a strict average and standard deviation are not
calculated. Rather than provide standard deviations to account for
the variability, confidence intervals are used. These are reported
in the table as the upper and lower limits in the parenthesis next
to AUC/day % TGI. Animal body weights were measured using an
Adventura Pro AV812 scale (Ohaus Corporation; Pine Brook, N.J.).
Graphs were generated using KaleidaGraph, version 3.6. Percent
weight change was calculated using the following formula: Group
percent weight change=(new weight-initial weight)/initial
weight).times.100
To appropriately analyze the repeated measurement of tumor volumes
from the same animals over time, a mixed modeling approach was used
(Pinheiro et al., Linear and Nonlinear Mixed Effects Models. (2008)
R package version 3.1-89). This approach addresses both repeated
measurements and modest dropouts due to any non-treatment-related
deaths of animals before the study end.
Cubic regression splines were used to fit a non-linear profile to
the time courses of log.sub.2 tumor volume at each dose level.
These non-linear profiles were then related to dose within the
mixed model. Tumor growth inhibition as a percentage of vehicle (%
TGI) was calculated as the percentage of the area under the fitted
curve (AUC) for the respective dose group per day in relation to
the vehicle, using the following formula: %
TGI=100.times.(1-AUC.sub.dose/AUC.sub.veh)
To determine the uncertainty intervals (UIs) for % TGI, the fitted
curve and the fitted covariance matrix were used to generate a
random sample as an approximation to the distribution of % TGI. The
random sample was composed of 1000 simulated realizations of the
fitted-mixed model, where the % TGI has been recalculated for each
realization. The reported UIs were the values for which 95% of the
time, the recalculated values of % TGI would fall in this region
given the fitted model. The 2.5 and 97.5 percentiles of the
simulated distribution were used as the upper and lower UIs.
Plotting was performed and generated using R, version 2.8.1 (R
Development Core Team 2008; R Foundation for Statistical Computing;
Vienna, Austria) and Excel, version 12.0.1 (Microsoft Corporation).
Data were analyzed using R, version 2.8.1, and the mixed models
were fit within R using the nlme package, version 3.1-89 (Pinheiro
et al., 2008).
FIGS. 12 & 13 show the in vivo activity of the anti-EGFR/c-met
bispecific antibody in KP4 pancreatic xenograft model and A431
epidermoid carcinoma xenograft model, respectively. The bispecific
antibody was able to inhibit the growth of the tumors in vivo for
both models as compared with control animals that received only the
vehicle as a treatment. The graphs indicate the tumor volume was
decreased by administration of the bispecific antibody with the
Linear Mixed Effects (LME) fitter tumor volume of 505 mm.sup.3 in
KP4 xenografts at 20 days after treatment compared to 1710 mm.sup.3
for the vehicle only arm and 328 mm.sup.3 in A431 xenografts after
20 days compared to 495 mm.sup.3 in the vehicle only control.
Overall there was a significant change in the AUC/day expressed as
% TGI. For the bispecific antibody treatment in the KP4 xenografts,
there was 85% tumor growth inhibition (TGI) and in the A431
xenograft models there was a 68% TGI.
Example 7
Heteromultimeric Protein Production Using CHO Cell Culture
This example illustrates the formation of heteromultimeric proteins
from a culture comprising two host CHO cell populations.
Half-antibodies containing either the knob or hole mutations were
generated in separate cultures by transiently expressing the heavy
and light chains using constructs and techniques well known in the
art. (See, for example, Ye et al., Biotechnol Bioeng. 2009 Jun. 15;
103(3):542-51.) Cells were cultured in 1 liter of media (see, for
example, Wong et al., J. Biol. Chem. 2007 282(31):22953-22963) and
harvested after 14 days.
Each half antibody was captured on a 5 mL MabSURE SELECT column.
The column was then washed with 10 column volumes (CV) of the
equilibration buffer followed by 10 CV of a sodium citrate low
conductivity buffer (equilibration buffer consisting of 50 mM TRIS
pH 8.0, 150 mM NaCl, 0.05% Triton X-100, 0.05% Triton X-114; low
conductivity wash buffer consisting of 25 mM Sodium Citrate pH
6.0). Each arm was eluted with 0.15 M Sodium Acetate pH 2.7.
Each half antibody was dialyzed into 50 mM TRIS pH 8.0, 150 mM
NaCl, 1 mM EDTA at a ratio of 1 to 300 at room temperature
overnight. Each arm was then centrifuged, filtered using 0.22
micron cellulose acetate filters and the two arms mixed together at
a ratio of 1 to 1 (the total concentration was less than 2 mg/mL).
The mixture was then processed one of two ways as follows:
Redox (with water bath): The mixture was then heated in a water
bath at 37.degree. C. After an hour, the redox mixture was removed
from the water bath and left to cool to room temperature. Once the
mixture reached room temperature, freshly prepared reducing agent,
dithiothreitol (DTT), was added to achieve a final concentration of
2 mM DTT. The mixture was left at room temperature for two hours,
then concentrated using Amicon Ultracell centrifugal filters (10K)
to 11 mg/mL and dialyzed into 50 mM TRIS pH 8.0, 150 mL NaCl
(1:300) overnight.
Redox (no water bath): The mixture was left at room temperature for
3 hours, after which freshly prepared reducing agent,
dithiothreitol (DTT), was added to achieve a final concentration of
2 mM DTT. The mixture was left at room temperature for two hours,
concentrated using Amicon Ultracell centrifugal filters (10K) to 11
mg/mL, and dialyzed into 50 mM TRIS pH 8.0, 150 mL NaCl (1:300)
overnight.
Following redox, the assembled material was purified on a 15 mL HIC
ProPac 10 column using a 20 CV gradient similar to the previous
section. The running buffer was 25 mM Potassium Phosphate, 0.7M
Ammonium Sulfate pH 6.5 and the elution buffer was 25 mM Potassium
Phosphate pH 6.5, 25% isopropanol. One mL fractions were collected
and peak fractions were separated by 4-20% Tris-Glycine SDS PAGE to
analyze purity and pooled accordingly. The pools were then
concentrated using Amicon Ultracell centrifugal filters (10K) to
around 1.5 mg/mL and dialyzed into 50 mM TRIS pH 8.0, 150 mM NaCl
and 0.22 .mu.m filtered.
The identity of each assembled bispecific was confirmed by Mass
Spectrometry. Purity was analyzed by 4-20% Tris-Glycine SDS PAGE
gel and bioanalyzer. Aggregate levels were determined by
SEC-MALS.
Results are shown in FIGS. 14-19. This example demonstrates that
bispecific antibodies can be produced using CHO host cells. One
skilled in the art will recognize that the method can be used to
produce other heteromultimeric proteins.
* * * * *